Jinal Shukla

Xieyang Guo

Claire Batisse

Charlotte Saint-André

Michael Webster

Arnaud Vanden Broeck

Alexandre Frechard

Corrine Crucifix

Gabor Papai

Anna Bonhoure

Camille Kostmann

Sirine Christophe Lotz

Leonid Andronov

Olga Kolesnikova

Adam Ben

Keywords: Transcription elongation & termination, NusG & NusA, single particles cryo-EM, dynamics of the RNAP

I would like to express my deepest

TABLE OF CONTENT

Table of figures

ii.

LA PAUSE DE LA TRANSCRIPTION

L'initiation était considérée comme l'étape cruciale de la transcription, et récemment nous avons mis en évidence que la phase d'élongation est aussi importante. La pause transcriptionnelle, arrêt temporaire du complexe d'ARNP pendant la transcription, est un mécanisme de régulation de l'expression des gènes très conservé (1,2). La pause est liée à la réalisation de nombreux processus cellulaires : le recrutement de facteurs de transcription (FT) à des temps donnés, la réparation de l'ADN, la fidélité de la transcription, la terminaison et le couplage de la polymérase avec le ribosome chez les bactéries. L'ARNP bactérien a tendance à faire une pause toutes les 100 paires de bases en moyenne, ces signaux de pause sont généralement codés dans la séquence d'ADN et sa durée peut être modulée par des FT.

iii. LES FACTEURS DE TRANSCRIPTION REGULANT LES PAUSES

Chez les procaryotes, le facteur NusG conjointement avec un autre factor, NusA, ont été identifiés au sein du complexe d'antiterminaison. Ils sont tous les deux essentiels pour les bactéries et impliqués dans la pause transcriptionnelle et la terminaison de la transcription (3,4). NusA stimule certaines type de pauses (3); en revanche, NusG supprime d'autres pauses pour augmenter la vitesse de la transcription (5). De manière surprenante, ces deux FT antagonistes, NusA et NusG, pourraient se lier simultanément à l'ARNP, selon des études de ChIP-on-chip à l'échelle du génome (6) et leur rapport molaire sur l'ARNP in vivo (7). Néanmoins, nous ne savons toujours pas comment ces deux FT exercent leur fonction au niveau mécanistique.

II. L'OBJECTIF

Les études structurales nous permettent de comprendre les mécanismes au niveau moléculaire. Précédemment, les chercheurs ont réussi à obtenir des structures de l'ARNP seule (8,9) et de complexes d'élongation canoniques (10,11). De plus en plus d'études sont réalisées sur l'ARNP en complexe avec des FT (12,13) car la plupart des ARNP sont liées aux FT in vivo (6).

Toutefois nous manquons d'informations sur la régulation des complexes d'élongation. Les propriétés biochimiques de la pause sont fortement conservées des procaryotes aux mammifères selon les études structurales (12,13). C'est pourquoi un modèle bactérien nous permet d'étudier la fonction essentielle des FT et leurs mécanismes fondamentaux sur la pause. La reconstruction du complexe NusG-ARNP ressemble à une conformation non pivotée (Figure 29). J'ai effectué un raffinement hétérogène dans CryoSPARC pour étudier la dynamique de la polymérase. Les particules ont été initialement triées en cinq classes (Figure 27). Deux classes (environ 40 % des particules) ne contenaient pas ou très peu de densité relative à NusG, mais différaient par la conformation de leur module pivotant (jusqu'à 2,4 degrés de rotation globale) (Figure 30). Ces deux classes représentent une ARNP-EC canonique sans aucune FT liée (ARNP-EC) et soulignent la liberté conformationnelle intrinsèque du module pivotant de l'ARNP. La classe de l'ARNP-EC non pivotée servira comme la référence pour mesurer l'ange de la rotation des autres structures (Figure 28). Trois classes contiennent une forte densité pour NusG et se différencient également par l'ampleur du pivotement (jusqu'à 3,3 degrés de rotation), et représentent NusG-EC (Figure 31).

Dans NusG-EC, la densité de l'ADN en amont de la bulle de transcription et le brin codant de l'ADN dans la bulle de transcription sont mieux définit dans NusG-EC par rapport à l'ARNP-EC (Figure 32). NusG prolonge une surface de l'ARNP chargée positivement qui est en contact avec le duplex d'ADN en amont de la bulle de transcription et du brin codant de l'ADN dans la bulle de transcription. Ceci a un effet stabilisateur en neutralisant la charge négative de l'ADN (Figure 33), ce qui permet

INTRODUCTION

Cellular function, metabolism and phenotype depend on precise decoding of genetic information. To achieve this goal, cells need to be extremely accurate in gene expression. Gene expression starts with transcription of DNA to RNA. This process involves a key enzyme, which is called RNA polymerase (RNAP). Its architecture is highly conserved throughout the three kingdoms of life [START_REF] Ebright | RNA polymerase: Structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II[END_REF]. Using simple models such as bacterial enzymes to understand the main transcription mechanisms can therefore contribute towards a better understanding of the same mechanisms in organisms of higher complexity as well as providing insights on evolution of key enzymes.

I. TRANSCRIPTION BY PROKARYOTIC RNA POLYMERASES

In prokaryotes, there is only one type of RNAP (Figure 1) [START_REF] Ebright | RNA polymerase: Structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II[END_REF].

In 1999 Zhang et al. solved the first structure of a cellular, multi-subunit polymerase from Thermus aquaticus [START_REF] Zhang | Crystal structure of thermus aquaticus core RNA polymerase at 3.3 å resolution[END_REF]. It consists of 5 subunits with the composition α2ββ'ω. The two α subunits contain two domains: a N-terminal domain (NTD) and a C-terminal domain (CTD), connected by a flexible linker. The two α-NTDs serve as an assembly platform for RNAP and interact with the two largest subunits called β and β' [START_REF] Ebright | The Escherichia coli RNA polymerase α subunit: structure and function[END_REF][START_REF] Gourse | UPs and downs in bacterial transcription initiation: The role of the alpha subunit of RNA polymerase in promoter recognition[END_REF][START_REF] Ishihama | Role of the RNA polymerase α subunit in transcription activation[END_REF]. The two α-CTDs interact with promoter DNA or with DNAbinding transcription factors (TFs) [START_REF] Ebright | The Escherichia coli RNA polymerase α subunit: structure and function[END_REF][START_REF] Gourse | UPs and downs in bacterial transcription initiation: The role of the alpha subunit of RNA polymerase in promoter recognition[END_REF][START_REF] Ishihama | Role of the RNA polymerase α subunit in transcription activation[END_REF]. The β and β' subunits form the catalytic core for RNA synthesis. They resemble two pincers of a crab claw, holding the DNA during transcription (Figure 1). The ω subunit promotes or stabilizes the β' subunit during or after assembly with the α2β assembly intermediate [START_REF] Mathew | The evolving story of the omega subunit of bacterial RNA polymerase[END_REF][START_REF] Minakhin | Bacterial RNA polymerase subunit ω and eukaryotic polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly[END_REF].

These five subunits together form the core enzyme. Transcription needs to be initiated at a promoter region, and the sigma factors (σ) have the ability to identify it. There are multiple σ factors in each species, and even more variety across species (Cook and Ussery, 2013;[START_REF] Li | Structural basis for transcription initiation by bacterial ECF σ factors[END_REF]Paget, 2015). The role of σ factors is to associate with the core enzyme and form holoenzyme that initiates specific gene expression programs under a variety of environmental conditions such as iron transport, metabolism (σ 19 (FecI)), or the heat shock response (σ 32 (RpoH)) (Cook and Ussery, 2013;[START_REF] Nakahigashi | Isolation and sequence analysis of rpoH genes encoding σ32 homologs from gram negative bacteria: Conserved mRNA and protein segments for heat shock regulation[END_REF]. Only one σ factor can associate with the core enzyme at a time. As a result, different holoenzymes initiate transcription for different sets of genes [START_REF] Li | Structural basis for transcription initiation by bacterial ECF σ factors[END_REF]. Initiation of transcription under normal growth conditions is done with the help of the factor σ 70 , called the "housekeeping" sigma factor or primary sigma factor [START_REF] Li | Structural basis for transcription initiation by bacterial ECF σ factors[END_REF]Paget, 2015). The first holoenzyme structures from T. thermophilus and T. aquaticus were solved in 2002 [START_REF] Murakami | Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution[END_REF][START_REF] Yokoyama | Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6. Å resolution[END_REF]. In 2013, Murakami et al. solved the first crystal structure of the E. coli RNAP holoenzyme (Murakami, 2013). More recently, single particle cryo-EM allowed the capture of intermediates during initiation using RNAP from various species (Boyaci et al., 2019).

II. INITIATION OF THE TRANSCRIPTION

Transcription initiation involves several steps. First, the closed complex forms when holoenzyme binds to double stranded and annealed DNA.

The σ-domain 4 (σ4) of σ 70 recognizes the "-35 element" (TTGACA), while σ-domain 2 (σ2) recognize the "-10 element" (TATAAT) at the promoter region (Figure 2) [START_REF] Bae | RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[END_REF][START_REF] Campbell | Structure of the bacterial RNA polymerase promoter specificity σ subunit[END_REF][START_REF] Feklistov | Structural basis for promoter -10 element recognition by the bacterial RNA polymerase σ subunit[END_REF][START_REF] Murakami | Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution[END_REF].

To form an open complex, the holoenzyme needs to change its conformation several times to properly unwind ~14 base pairs (bp) of the double strain DNA near the "-10 element" with a bending angle, and position the DNA in the active site (Boyaci et al., 2019;[START_REF] Browning | Local and global regulation of transcription initiation in bacteria[END_REF][START_REF] Buc | Kinetics of Open Complex Formation between Escherichia coli RNA Polymerase and the lac UV5 Promoter. Evidence for a Sequential Mechanism Involving Three Steps[END_REF][START_REF] Kapanidis | Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism[END_REF][START_REF] Lisica | An RNA dependent nucleoside triphosphate phosphohydrolase (ATPase) associated with rho termination factor[END_REF][START_REF] Nogales | Structural Insights into the Eukaryotic Transcription Initiation Machinery[END_REF][START_REF] Plaschka | Cooperation between translating ribosomes and RNA polymerase in transcription elongation[END_REF][START_REF] Roe | Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λPR promoter[END_REF][START_REF] Saba | Mechanism of bacterial transcription initiation: RNA polymerase -Promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis[END_REF]. Once the open complex is properly formed and stable enough, initial RNA synthesis occurs starting at the transcription start site (TSS or +1 template position). rNTPs diffuse to the RNAP active site through the secondary channel, and the newly formed RNA chain emerges from the RNA exit channel. To translocate in the forward direction in this initially transcribing complex, RNAP pulls the downstream DNA into the active site, extends the transcription bubble at the downstream edge but does not move relative to the upstream position on the DNA [START_REF] Kapanidis | Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism[END_REF]. This is called 'DNA-scrunching' (Figure 3).

However, 'scrunching' introduces stress in the RNAP. The more RNAP extends the DNA bubble, the more stress is created. To ease the stress, either RNAP needs to backtrack (i.e. back-translocate), extrude the RNA into the secondary channel accompanied with RNA cleavage, and release the short transcript (abortive transcript) [START_REF] Goldman | Direct detection of abortive RNA transcripts in vivo[END_REF][START_REF] Zuo | Crystal structures of the e.coli transcription initiation complexes with a complete bubble[END_REF], or σ4 breaks its interaction with the promoter DNA and RNAP escapes to enter the elongation phase [START_REF] Zuo | Structural Insights into Transcription Initiation from De Novo RNA Synthesis to Transitioning into Elongation[END_REF].

It has also been proposed, that 'DNA-scrunching' of the template-DNA (t-DNA) generates a force that helps RNAP to escape the promotor and enter elongation by breaking the contact between σ-domain 3 (σ3) and the core enzyme [START_REF] Zuo | Structural Insights into Transcription Initiation from De Novo RNA Synthesis to Transitioning into Elongation[END_REF]. This action also removes a loop of σ3 that blocks the RNA exit channel, which opens up the path for the [START_REF] Kapanidis | Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism[END_REF] nascent RNA [START_REF] Zuo | Structural Insights into Transcription Initiation from De Novo RNA Synthesis to Transitioning into Elongation[END_REF]. The affinity between the core enzyme and σ will be reduced when RNAP enters the elongation state, which is why this is accompanied by a release of σ [START_REF] Travers | CycliC Re-use of the RNA Polymerase Sigma Factor[END_REF]. However, it has also been shown that σ can stay associated with RNAP during the early phase of elongation [START_REF] Bar-Nahum | Isolation and characterization of σ70-retaining transcription elongation complexes from Escherichia coli[END_REF].

III. ELONGATION OF TRANSCRIPTION

Once RNAP enters the elongation phase, the size of the transcription bubble is maintained at a size of roughly 10 to 12 bp along with an RNA-DNA hybrid length of 9 to 10 bp [START_REF] Kent | Maintenance of RNA-DNA hybrid length in bacterial RNA polymerases[END_REF][START_REF] Kireeva | Translocation by multi-subunit RNA polymerases[END_REF].

The transcription bubble is covered by the two subunits β and βʹ, which wrap around each other to create a cavity called the primary channel (Figure 4 C-D) [START_REF] Bar-Nahum | The Mechanisms of Substrate Selection, Catalysis, and Translocation by the Elongating RNA Polymerase[END_REF]. Catalysis in the active centre takes place in this channel. A long protein helix spans the active centre, called the bridge helix (BH) (Figure 4 C). It adopts two forms: straight and bent, and it has been hypothesized that the bending of the bridge helix could help the translocation of the transcribing RNAP and in melting the downstream DNA [START_REF] Bar-Nahum | The Mechanisms of Substrate Selection, Catalysis, and Translocation by the Elongating RNA Polymerase[END_REF][START_REF] Hein | The bridge helix coordinates movements of modules in RNA polymerase[END_REF][START_REF] Kireeva | Translocation by multi-subunit RNA polymerases[END_REF]. The trigger loop (TL) is found close to the BH and also near the active site (Figure 4 E-F), and is an essential element for catalysis [START_REF] Zhang | Role of the RNA polymerase trigger loop in catalysis and pausing[END_REF]. In presence of the cognate substrate, it folds into two helices (Figure 4 F), and together with the BH forms a three-helix bundle (THB) (Figure 4 E) [START_REF] Vassylyev | Structural basis for substrate loading in bacterial RNA polymerase[END_REF][START_REF] Wang | Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis[END_REF]. The secondary channel ( [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF][START_REF] Hein | RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement[END_REF]. Behind the β-lobe and the shelf, between subunit ω and the β flap domain, is the RNAP core module which appears to be the structurally most stable part of RNAP (Figure 4 B). It is mainly formed by the two α subunits and large parts of the βsubunit (except for β-lobe and protrusion). Different elements, modules, domains and subunits of RNAP are labelled in the figure in A-H. E and F adopted from [START_REF] Bar-Nahum | The Mechanisms of Substrate Selection, Catalysis, and Translocation by the Elongating RNA Polymerase[END_REF][START_REF] Zhang | Role of the RNA polymerase trigger loop in catalysis and pausing[END_REF].

i.

NUCLEOTIDE ADD ITIO N CYCLE

During elongation the nucleotide addition cycle is repetitively carried out. At the beginning of the cycle, the primary channel contains 9 bp of RNA-DNA hybrid with an unpaired template DNA base at the A site (Acceptor site, or i+1 site). This is called the post-translocated state (Figure 5). rNTP substrates diffuse into the RNAP active site through the secondary channel arriving at the A site (i+1 site). If a cognate rNTP complementary to the template DNA binds in the A site, the TL folds into the TH to stabilize the newly bound rNTP for catalysis. Two Mg2+ ions (Mg2+ I and Mg2+ II) stabilize the rNTP, and Mg2+ I lowers the pKa of the 3'-OH of the 3'-terminal nucleotide in the RNA chain in the P site (Product site, also called i site) (Figure 5). Catalysis occurs when the 3'-OH attacks the α-phosphate of the rNTP through a nucleophilic substitution (SN2) reaction, releasing a pyrophosphate [START_REF] Steitz | A mechanism for all polymerases[END_REF].

Once the chemistry step has finished, Mg2+ II dissociates from the active site bound to the leaving pyrophosphate through the secondary channel. The RNA-DNA hybrid now contains 10 bp in what is called the pre-translocated state (Figure 5). RNAP needs to translocate forward by one nucleotide, position the next template DNA base in the A site, and free the rNTP binding site. The interaction between the 3'-OH and Mg2+

I becomes strong [START_REF] Bar-Nahum | The Mechanisms of Substrate Selection, Catalysis, and Translocation by the Elongating RNA Polymerase[END_REF]. This is important for positioning the RNA 3ʹ-end in the P site. RNAP must maintain the size of the transcription bubble during translocation. This means during translocation one base pair of the downstream DNA has to be melted, one base pair at the upstream end of the RNA-DNA hybrid is separated and a new base pair between template and non-template DNA forms as the upstream DNA rewinds. With the 3'-OH of the RNA positioned at the P site (or i site), leaving the A site (or i+1 site) empty for the next incoming rNTP, the hybrid is now in a post-translocated state with 9 bp concluding the nucleotide addition cycle (Figure 5). Some RNAP modules are involved during the translocation process. The fork loop-2 module (β533 -548) is found near the BH and between the downstream DNA and hybrid. It acts as a barrier for the entrance of the downstream DNA into the active site, helping the downstream DNA to unwind and to properly position the template DNA in the active centre [START_REF] Gnatt | Structural basis of transcription: An RNA polymerase II elongation complex at 3.3 Å resolution[END_REF][START_REF] Vassylyev | Structural basis for substrate loading in bacterial RNA polymerase[END_REF]. At the upstream edge of the hybrid, the lid loop stacks on the terminal RNA-DNA hybrid base pair and helps to separate the RNA strand from the template DNA [START_REF] Naryshkina | The Role of the Largest RNA Polymerase Subunit Lid Element in Preventing the Formation of Extended RNA-DNA Hybrid[END_REF]. Switch-3 (β1248 -1269 interacts with the first single-stranded RNA base in the exit channel by forming a hydrophobic pocket [START_REF] Kent | Maintenance of RNA-DNA hybrid length in bacterial RNA polymerases[END_REF]. This is how RNAP maintains the same size of the RNA-DNA hybrid (9 or 10 bp), while transcribing along the DNA and switching between the pre-and post-translocated states. RNAP is highly processive and stays bound to the DNA until it finishes transcribing a single unit (gene). Consequently, a termination step is needed to actively dissociate the RNAP, DNA, and nascent RNA.

IV. TERMINATION OF TRANSCRIPTION

The dissociation of the elongation complex (EC) from DNA requires an active mechanism to destabilize the complex and terminate at a specific position. Intrinsic termination and ρ-dependent termination have evolved as the two dominant pathways in prokaryotic organisms (Park and Roberts, 2006).

i. INTRINSIC TERMINATION

The intrinsic termination pathway can occur by itself, does not depend on additional protein factors, and does not require energy. Termination occurs at a region rich in guanines and cytosines (GC) followed by a poly-U sequence [START_REF] Wilson | An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA[END_REF]. The GC-rich sequence is partially or fully self-complementary and forms an RNA hairpin structure in the RNA exit channel of RNAP. RNAP stops RNA synthesis and halts at the end of the poly-U stretch. The hairpin may prevent RNAP back-translocation and locks the EC on the weak hybrid (poly-U) region [START_REF] Komissarova | Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination[END_REF]. The hairpin stem may also invade the RNA-DNA hybrid and thus destabilize it further. Furthermore, the weak poly-U sequence makes the RNA-DNA hybrid unstable and likely helps in complex dissociation [START_REF] Gusarov | The mechanism of intrinsic transcription termination[END_REF]. Termination requires a tight coordination of RNA hairpin formation and RNAP position on the template, as both the RNA hairpin and poly-U sequence are indispensable for the destabilization and dissociation of the EC.

However, the complete mechanism remains unknown. The termination efficiency is affected by the size of the stem, the sequence of the stem and the length of the loop [START_REF] Cheng | Functional importance of sequence in the stem-loop of a transcription terminator[END_REF][START_REF] Reynolds | Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators[END_REF][START_REF] Wilson | An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA[END_REF].

ii.

RHO-DEPENDENT TERMINATION

This alternative termination pathway requires the binding of protein factors. One termination factor termed ρ, is highly conserved and nearly ubiquitous in bacteria [START_REF] Opperman | Phylogenetic analysis of sequences from diverse bacteria with homology to the Escherichia coli rho gene[END_REF]. It is essential in E. coli, where nearly 20% of protein-coding genes (Peters et al., 2009) require ρ-dependent termination. It plays a role in the termination of one third of tRNA operons and several small RNAs (Peters et al., 2009).

ρ looks like a doughnut that consists of six identical monomers (Figure 6). It is an adenosine triphosphate (ATP)-dependent (Lowery Goldhammer and Richardson, 1974) RNA-DNA helicase [START_REF] Brennan | Transcription termination factor rho is an RNA-DNA helicase[END_REF]. ρ-utilization (rut) sites serve as ρ-binding elements, and are approximately 80-90 nt long [START_REF] Chen | Sequence elements essential for rhodependent transcription termination at lambda tR1[END_REF][START_REF] Mcswiggen | Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies[END_REF]. The rut site is a long sequence without any secondary structure, but rich in cytosines and poor in guanines [START_REF] Allfano | A consensus motif common to all rho-dependent prokaryotic transcription terminators[END_REF][START_REF] Morgan | RNA sequence and secondary structure requiresments for rho-dependent transcription termination[END_REF]. ρ has the highest affinity for poly-cytidine RNA sequences, and the ATPase activity is mostly activated by the cytidine residues (Lowery Goldhammer and Richardson, 1974;[START_REF] Richardson | Rho-dependent termination and ATPases in transcript termination[END_REF]. However, there is no consensus sequence of the rut site, and various C-rich RNA sequences can stimulate ρ [START_REF] Richardson | Rho-dependent termination and ATPases in transcript termination[END_REF].

Termination after a rut site does not appear to occur at a specific site but over a range of positions. For example, λtR1 has originally been reported to contain three major sites of termination [START_REF] Lau | Transcription terminates at λt(R1) in three clusters[END_REF] while more recent biochemical work divided the region into an early and a late termination region [START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF]. However, it has been suggested based on RNA-seq data, that in vivo transcripts terminated by ρ might undergo trimming on their 3'-ends until a stable secondary structure (such as a hairpin) is reached as a means to stabilize the transcript against 3'-5'exonucleases [START_REF] Dar | High-resolution RNA 3-ends mapping of bacterial Rhodependent transcripts[END_REF]. [START_REF] Ray-Soni | Mechanisms of Bacterial Transcription Termination: All Good Things Must End[END_REF][START_REF] Richardson | Rho-dependent termination and ATPases in transcript termination[END_REF]. However, biochemical and recent structural studies showed that ρ is more likely to interact first with RNAP and then screen for the rut RNA sequence [START_REF] Epshtein | An allosteric mechanism of Rho-dependent transcription termination[END_REF][START_REF] Hao | Pre-termination Transcription Complex: Structure and Function[END_REF][START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. ρ appears to be able and inactivate the EC in its 'open-ring' state [START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF].

b) B I N D I N G , C O N F O R M A T I O N A L C HA N G E S , A N D T R A N S L O C A T I O N O F R
Otherwise, the newly transcribed rut RNA makes a detour on each NTD of the monomer of ρ and passes through the central hole constituted by the CTD of ρ. ρ therefore isomerizes into a closed-ring state and translocate the RNA.

c) C O M P L E X D I S S O C I A T I O N

The mechanism triggering dissociation of the EC from the DNA remains unclear. The traditional models of transcription termination proposed that after ρ encounters the paused RNAP, ρ might either pull the RNA from the EC or push RNAP forward [START_REF] Ray-Soni | Mechanisms of Bacterial Transcription Termination: All Good Things Must End[END_REF][START_REF] Richardson | Rho-dependent termination and ATPases in transcript termination[END_REF]. But the last recent studies showed that at the rut termination level, ρ induces the clamp opening of a paused RNAP and thus may release the nucleic acid from EC [START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. All mechanisms converged into one goal, trapping and destabilization of the EC and releasing nucleic acids to accomplish the dissociation.

Once the EC detaches from the DNA and releases the RNA, RNAP is then free to re-associate with a σ factor and initiate another round of transcription [START_REF] Helmann | RNA polymerase: A nexus of gene regulation[END_REF][START_REF] Mooney | Sigma and RNA polymerase: An onagain, off-again relationship?[END_REF].

V. THE REGULATION OF THE TRANSCRIPTION

As unicellular organisms, bacteria are directly exposed to the environment, where conditions such as temperature, nutrients, oxygen levels, pH and osmolarity all vary. Bacteria can also be subjected to chemical stress and physical stress. Prominent examples are antibiotics or heat shock. To adapt to all these conditions, gene expression must occur to up-or down-regulate a selected set of genes at various moments, and the RNA copy-number must reflect what the organism needs. Furthermore, more temporal control will be exerted by intraand extracellular signals once the selected genes are active. For each protein-coding gene, there are specific combinations of transcription factors (TFs). These TF's regulate gene expression at each step of transcription, a process that is very complex and vital for all living organisms. Thus, the regulation of transcription can be modulated by TFs in the forms of RNA or proteins, but signals to modulate transcription can also be encoded directly by the DNA.

i. TRANSCRIPTIONAL PAUSING

Transcription elongation is not a smooth process but frequently interrupted by transcriptional pauses, which are temporary halts in the nucleotide addition cycle. Transcriptional pausing is a widespread regulatory mechanism, employed during initiation, early and late elongation and also termination of transcription [START_REF] Helmann | RNA polymerase: A nexus of gene regulation[END_REF]Landick, 2006;[START_REF] Nandymazumdar | Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins -Shifting shapes and paradigms[END_REF]. It is also crucial for transcriptional fidelity [START_REF] Kwak | Control of Transcriptional Elongation[END_REF].

RNAP pauses while attempting to escape from the promoter during initiation as described above to respond to the 'scrunching' stress.

When RNAP reaches the end of transcripts, the termination pathways compete with elongation. Slowing down the elongation rate by pausing could favour termination efficiency and help to initiate the termination process [START_REF] Gusarov | The mechanism of intrinsic transcription termination[END_REF][START_REF] Yarnell | Mechanism of intrinsic transcription termination and antitermination[END_REF]. When the rNTP concentration is limited in vitro, the transcription rate slows down and the termination efficiency increases. This "kinetic coupling" model indicates a correlation between transcriptional pausing and termination [START_REF] Jun | Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho[END_REF]. During ρ-dependent termination, RNAP pauses at all the terminators, but can escape the pause and continue elongation before ρ initiates termination [START_REF] Lau | Transcription terminates at λt(R1) in three clusters[END_REF].

Both the RNA hairpin and the poly-U sequence in the intrinsic termination process induce pausing, which in turn favours termination.

Pausing may also facilitate the proper folding of the RNA terminator hairpin in case of intrinsic termination sites. pause sites genome-wide and across well-transcribed genes of E. coli [START_REF] Larson | A pause sequence enriched at translation start sites drives transcription dynamics in vivo[END_REF]. Therefore, RNAP pauses on average every 100 base pairs for brief periods of time (on the order of seconds) [START_REF] Larson | A pause sequence enriched at translation start sites drives transcription dynamics in vivo[END_REF][START_REF] Vvedenskaya | Interactions between RNA polymerase and the "core recognition element" counteract pausing[END_REF]. As briefly explained, pausing has multiple functions in gene expression: i) it facilitates the proper folding of nascent RNA transcripts [START_REF] Pan | Folding of a large ribozyme during transcription and the effect of the elongation factor NusA[END_REF]; ii) it enables the interaction or recruitment of TFs at a given time [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF]; iii) it allows the coupling between transcription and translation [START_REF] Yanofsky | Attenuation in the control of expression of bacterial operons[END_REF] in bacterial attenuators; and iv) it is involved in transcriptional fidelity to correct mis-incorporated rNTPs [START_REF] Erie | Multiple RNA polymerase conformations and GreA: Control of the fidelity of transcription[END_REF].

Pause signals are often encoded in the underlying DNA sequences.

When RNAP encounters a pause site, a certain fraction of the molecular ensemble enters the elemental paused state (this is referred to as the pause efficiency). A subset of these RNAP molecules may enter a longerlived pause, while the rest escapes the pause and resumes transcription.

Two broad categories of pausing have been described to drive RNAP from elemental paused states into long-lived pauses, termed class I and class II.

b) C L A S S I P A U S E : HAIRP I N -S T A B I L I Z ED P A U S I N G

In the class I pause, the long-lived pause of RNAP is stabilized by the nascent RNA that forms a hairpin in the RNA exit channel. It repositions the hybrid at the active site and inactivates RNAP. The class I pause signal is mostly found in the leader region of amino acid biosynthetic operons, where it coordinates transcription with translation [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF]. One of the best-characterized class I pauses, termed the his-pause, is located in the leader region of the hisoperon, as its name implies. It controls the biosynthesis of histidine by synchronizing RNAP and ribosome movement during attenuation [START_REF] Chan | The Salmonella typhimurium his operon leader region contains an RNA hairpin-dependent transcription pause site. Mechanistic implications of the effect on pausing of altered RDNA hairpins[END_REF]. This pause is strongly enhanced and stabilized by the formation of a nascent RNA hairpin in the RNA exit channel of RNAP [START_REF] Toulokhonov | Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins[END_REF]. The structure of the RNA is more important than the sequence itself for pausing enhancement [START_REF] Chan | Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin[END_REF][START_REF] Chan | Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways[END_REF][START_REF] Toulokhonov | Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins[END_REF]. The structure of the RNA hairpin constitutes a 5 base pair stem and an 8 nucleotide loop and it is separated from the pause site (3' end of RNA) by 11 base pairs. The region between the RNA 3'-end and the RNA hairpin, the nucleotide in the active site, and the first 14 bp of downstream DNA also affect the his-pause efficiency [START_REF] Chan | Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways[END_REF]. with the active site. The likelihood of forward translocation and cleavage seem to be a function of the length of the backtracked RNA [START_REF] Lisica | An RNA dependent nucleoside triphosphate phosphohydrolase (ATPase) associated with rho termination factor[END_REF]. RNAP itself can catalyse hydrolysis of the RNA through an intrinsic endo-nucleolytic activity, and binding of TFs can increase the rate of cleavage (Borukhov et al., 1993). One wellcharacterized class II pause is called the ops-pause (ops = operon polarity suppressor) [START_REF] Bae | RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[END_REF]. It is triggered by a short sequence located in the early transcribed but non-translated regions of horizontally acquired operons including ones encoding the biosynthesis of polysaccharides [START_REF] Bae | RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[END_REF]. There are 15 candidate ops-sites,

which have been identified in E. coli. 12 of them encode enzymes for the synthesis of extra-cytoplasmic macromolecules, and two of them are located on the upstream end of the pheP and rfaQ genes [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF]. The ops-pause permits the recruitment of the TF RfaH, which is an auxiliary protein that suppresses pausing and premature termination of transcription [START_REF] Bae | RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[END_REF],

and guarantees long transcripts of up to 20 kb [START_REF] Bae | RfaH and the ops element, components of a novel system controlling bacterial transcription elongation[END_REF][START_REF] Bailey | In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element[END_REF][START_REF] Leeds | Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism[END_REF].

d) T H E C O N S E N S U S S E Q U E N C E O F T R A N S C R I P T I O N A L P A U S I N G .
Pausing depends on two factors: 1) the fraction of RNAP that enters the paused state (pause-efficiency), and 2) the duration spent by RNAP at the pause site (the residence time, often expressed as pause half-life).

In 2014, the Greenleaf, Landick, and Weissman lab as well as the Nickels and Ebright lab identified a consensus pause sequence from genome wide studies in E. coli [START_REF] Larson | A pause sequence enriched at translation start sites drives transcription dynamics in vivo[END_REF][START_REF] Vvedenskaya | Interactions between RNA polymerase and the "core recognition element" counteract pausing[END_REF].

The closer the DNA sequence matches the consensus sequence, the stronger the pausing will be and the higher the fraction of RNAP entering the pause (the higher the pause efficiency). The most important features of the consensus pause site appear to be the RNA sequence at positions -10 and -11 (most often G), the identity of the RNA 3'-end (most frequently a U or C), and the next incoming nucleotide (most frequently a GTP or ATP). The consensus pause sequence can thus be summarized as G-11G-10Y-1G+1 (Figure 8) [START_REF] Larson | A pause sequence enriched at translation start sites drives transcription dynamics in vivo[END_REF][START_REF] Vvedenskaya | Interactions between RNA polymerase and the "core recognition element" counteract pausing[END_REF]. This combination of bases probably slows down translocation and thus the nucleotide addition cycle, and increases the chance for RNAP to enter a long-lived pause [START_REF] Gusarov | The mechanism of intrinsic transcription termination[END_REF][START_REF] Komissarova | RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA[END_REF][START_REF] Nudler | Coupling between transcription termination and RNA polymerase inchworming[END_REF][START_REF] Wang | Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex[END_REF]. In addition, the sequence of the downstream DNA might also influence pausing although how this is might be achieved is less clear [START_REF] Palangat | Downstream DNA selectively affects a paused conformation of human RNA polymerase II[END_REF].

Certain TFs influence pause duration and the pausing frequency. For example, NusA stimulates class I pauses by interacting with the RNA hairpin and stabilizing the RNAP in the non-active conformation [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF][START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]Landick, 2006). And the ops-pause, a class II pause, can be prolonged by RfaH, which stabilizes the EC in a backtracked paused conformation by interacting with the non-template DNA [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF]; Kang et

VI. TRANSCRIPTION FACTORS NUSA AND NUSG

Pausing can be modulated by various auxiliary proteins in different organisms. In my thesis I focussed on two TFs called NusA and NusG.

They are both essential general transcription elongation factors and are present in prokaryotes and archaea [START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF][START_REF] Nandymazumdar | Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins -Shifting shapes and paradigms[END_REF][START_REF] Shibata | Crystal structure and RNA-binding analysis of the archaeal transcription factor NusA[END_REF].

Especially, NusG is the only universally conserved TF present in every kingdom of life. In Archaea and yeast, NusG is named Spt5, and in humans it is a component of DSIF [START_REF] Nandymazumdar | Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins -Shifting shapes and paradigms[END_REF]. Both NusA and NusG are highly conserved, and scientists have studied their functions and structures for more than 40 years. However, they are both multifunctional TFs affecting the rate of elongation and regulating transcription termination, making research on them more complicated. NusG was discovered along with rpoB and rpoC (E. coli RNAP β and β' subunits) in the λrifd cluster in 1979 [START_REF] Downing | Sequence and transcriptional pattern of the essential Escherichia coli secE-nusG operon[END_REF][START_REF] Yamamoto | Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome[END_REF]. NusA was discovered in 1974-1975 as part of the anti-termination complex manipulated by the protein N of phage lambda [START_REF] Friedman | Genetic characterization of a bacterial locus involved in the activity of the N function of phage λ[END_REF], and thus it was found that NusA stimulates the β-galactosidase synthesis [START_REF] Kung | Purification and properties of a soluble factor required for the deoxyribonucleic acid directed in vitro synthesis of β galactosidase[END_REF]. They were renamed as Nus factors. 'Nus' is an abbreviation and stands for 'N-utilizing substance'. The anti-termination complex contains not only NusA and NusG, but also two other Nus factors: NusB, and NusE. These

TFs, together with suhB and s4, also promote anti-termination in bacterial rrn operons [START_REF] Huang | Structure-Based Mechanisms of a Molecular RNA Polymerase/Chaperone Machine Required for Ribosome Biosynthesis[END_REF]. Different combinations of Nus factors are able to modify the properties of transcribing RNAP and realize different regulations in gene expression.

i. III.1 ROLE OF NUSA

NusA alone is mostly known to stabilize class I pauses. However, NusA also accelerates ribozyme folding by more than 10-fold by stimulating pausing of E. coli RNAP [START_REF] Pan | Folding of a large ribozyme during transcription and the effect of the elongation factor NusA[END_REF]. Besides this, NusA enhances intrinsic termination [START_REF] Schmidt | nusA Protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites[END_REF]. In particular, weak terminator hairpins depend on the presence of NusA according to genome wide studies (Mondal et al., 2016). It is involved in DNA damage repair and stress-induced mutagenesis (Cohen and Walker, 2010;Cohen et al., 2010).

a) I II. 1.1 STR U C T U R E D E S C R I P T I O N O F N U S A
NusA is a 55-kDa monomeric RNA binding protein in E. coli. We can hnRNP K, where it was first discovered) [START_REF] Gopal | Crystal structure of the transcription elongation/antitermination factor NusA from Mycobacterium tuberculosis at 1.7 Å resolution[END_REF]Worbs et al., 2001). The AR1 and AR2 domains (comprising the NusA-CTD), are not present in all NusA homologues [START_REF] Eisenmann | The E. coli NusA carboxy-terminal domains are structurally similar and show specific RNAPand λN interaction[END_REF]. The NusA-NTD and the RNA binding domains of NusA are highly conserved across different species. The 'βααβ' motif in the KH1 and KH2 provide a positively charged surface, which facilitates nucleic acid-binding that is also commonly found in many nucleic acid-binding proteins. [START_REF] Gopal | Crystal structure of the transcription elongation/antitermination factor NusA from Mycobacterium tuberculosis at 1.7 Å resolution[END_REF]Worbs et al., 2001). charged, making it a good candidate for the globular head domain of the NusA, which is negatively charged [START_REF] Shin | Crystal Structure of NusA from Thermotoga Maritima and Functional Implication of the N-Terminal Domain[END_REF]. And the interaction between FTH and NusA is mediated through hydrophobic contacts between a pocket in NusA-NTD and the FTH, which inserts into the pocket [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]. Another contact point between NusA and RNAP is through the KH1 and KH2 domains, which bind the RNAP ω subunit. Previous studies demonstrated that the intermediate domains (S1, KH1, and KH2) are RNA-binding domains [START_REF] Beuth | Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ[END_REF]. To prevent binding of free NusA to RNA, the NusA-CTD (AR1 and AR2) acts as an auto-inhibitory domain masking the RNA binding sites. This autoinhibitory effect is released when NusA-CTD binds to the α1-CTD of RNAP or to phage 𝝀 protein N [START_REF] Beuth | Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ[END_REF]Mah et al., 2000;[START_REF] Said | Structural basis for λN-dependent processive transcription antitermination[END_REF]. Overall, NusA exhibits high intrinsic flexibility and it also moves relative to RNAP because most contact points are tethered to the EC through flexible linkers. NusA rotates by binding the FTH and using it as the anchor and pivot point. It has been proposed that its flexibility is important for it to adapt to its various roles during transcription elongation and (anti-)termination [START_REF] Said | Structural basis for λN-dependent processive transcription antitermination[END_REF].

c) I II. 1.3 M E C HA N I S M O F P A U S I N G R EG U L A T I O N W I T H N U S A
Previous studies showed that the NusA-NTD is sufficient to stimulate the his-pause [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF][START_REF] Ha | The NusA N-Terminal Domain Is Necessary and Sufficient for Enhancement of Transcriptional Pausing via Interaction with the RNA Exit Channel of RNA Polymerase[END_REF][START_REF] Hein | RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement[END_REF] [START_REF] Ha | The NusA N-Terminal Domain Is Necessary and Sufficient for Enhancement of Transcriptional Pausing via Interaction with the RNA Exit Channel of RNA Polymerase[END_REF]. The NTD of NusA is sufficient to enhance pausing, and deletion of the FTH, which binds the NusA-NTD, abolishes pausing [START_REF] Toulokhonov | Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins[END_REF]. NusA presumably enhances intrinsic termination in an analogous way by favouring terminator hairpin folding and stabilizing RNAP in a paused state, which is a prerequisite for termination. [START_REF] Gusarov | The mechanism of intrinsic transcription termination[END_REF][START_REF] Kassavetis | Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro[END_REF] ii.

III.2 ROLE OF NUSG

NusG factors are classified into two categories: one category includes the ubiquitous housekeeping NusG proteins interacting with RNAP during elongation, and the other one includes specialized NusG paralogs (NusG SP ) modulating a subset of genes or particular operons [START_REF] Belogurov | Functional specialization of transcription elongation factors[END_REF]. Even housekeeping proteins often appear to have contradictory roles. In contrast to NusA, which mostly assists in RNA folding and enhances RNA-induced pausing during elongation or at intrinsic terminators, the role of NusG is much more complicated. For example, in Bacillus subtilis, NusG induces pausing, similarly to NusA.

NusG recognizes a TTNTTT motif in the non-template DNA strand in the transcription bubble, and this causes RNAP to pause on average once per 3 kb [START_REF] Yakhnin | NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro[END_REF]. However, NusG might suppress elongation complex pausing in regions without the TTNTTT motif. NusG from Thermus thermophilus also decreases the transcription rate (Sevostyanova and Artsimovitch 2010). However, in E. coli, NusG reduces RNAP pausing and stimulates ρ-dependent termination in vitro [START_REF] Burns | Activation of Rhodependent transcription termination by NusG: Dependence on terminator location and acceleration of RNA release[END_REF].

a) I II. 2.1 STR U C T U R E D E S C R I P T I O N O F N U S G
NusG is a small monomeric protein of roughly 20 kDa. It is composed of From biochemical studies, we know that the NusG-NTD domain is necessary and sufficient to prevent pausing (Mooney et al., 2009a). In [START_REF] Kireeva | RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate[END_REF][START_REF] Turtola | NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble[END_REF]. In the RNAP core (i.e.

factor-free) transcription EC this particular base pair is disordered, indicating its structural flexibility and it is the first one that needs to be melted for backtracking to take place (Kang et al., 2017). Second, they proposed that the binding of NusG and RfaH near RNAP active site cleft is incompatible with the swivelled state of RNAP, which is considered to be a paused conformation. This anti-swivelling effect is only demonstrated with the RfaH-NTD, but it is believed to be similar for NusG because it would supposedly clash with the swivelled conformation of RNAP (Kang et al., 2018a). Third, the RfaH-NTD stabilizes the nt-DNA with the ops sequence in the transcription bubble, and this specific interaction results in a formation of a nt-DNA hairpin.

However, no direct observation has been made of the nt-DNA in a hairpin form in the NusG-opsEC, but Kang et al. proposed that NusG might have a similar effect. Meanwhile, other studies suggested that NusG and Spt5 prevent the EC from adopting non-productive conformations by constraining the path of the nt-DNA [START_REF] Crickard | Biochemical analysis of yeast suppressor of Ty 4/5 (Spt4/5) reveals the importance of nucleic acid interactions in the prevention of RNA polymerase II arrest[END_REF][START_REF] Mazumdar | RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes[END_REF] or shortening the nt-DNA strand in an artificial EC [START_REF] Nedialkov | Locking the nontemplate DNA to control transcription[END_REF]. Compared to RfaH, NusG is less sequence-specific [START_REF] Mooney | Regulator Trafficking on Bacterial Transcription Units In Vivo[END_REF], which might explain why the three points mentioned above are better demonstrated in the RfaH-opsEC complex than in the NusG-opsEC. Indeed, RfaH has much higher affinity to the ops sequence compared to NusG.

VII. THE COUPLING OF TRANSCRIPTION AND TRANSLATION IN BACTERIA

In Prokaryotes, transcription and translation occur concurrently and in close spatial proximity. In 1964, Marshall Nirenberg's lab, based on biochemical evidence, proposed that transcription and translation might be coupled, which has regulatory effects on gene regulation [START_REF] Byrne | THE IN VITRO FORMATION OF A DNA-RIBOSOME COMPLEX[END_REF]. This suggestion was consistent with electron micrographs of actively expressed bacterial genes visualized in so-called Miller spreads. The micrographs show particles of sizes comparable to RNAP and the ribosome close to each other [START_REF] Miller | Visualization of bacterial genes in action[END_REF].

Transcription and translation also appear to occur at comparable rates in vivo, which argues for a kinetic coupling of the two processes.

However, this is still controversial, available data points both ways, and it appears there are species dependent differences [START_REF] Johnson | Structural basis of transcription arrest by coliphage HK022 nun in an escherichia coli rna polymerase elongation complex[END_REF][START_REF] Plaschka | Cooperation between translating ribosomes and RNA polymerase in transcription elongation[END_REF][START_REF] Zhu | Disruption of transcription-translation coordination in Escherichia coli leads to premature transcriptional termination[END_REF].

As explained before, NusG is composed of an NTD and a CTD, which are connected with a flexible linker. NusG recruits other TFs, such as termination factor ρ, to RNAP through its CTD. However, it has been suggested based on in vitro and in vivo data that NusG is able to form a physical connection between RNAP and the ribosome. This model proposed that the NusG-NTD binds RNAP, while the NusG-CTD binds ribosomal protein uS10 [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF][START_REF] Saxena | Escherichia coli transcription factor NusG binds to 70S ribosomes[END_REF].

Interestingly, uS10 serves dual functions as ribosomal protein in the 30S subunit and as transcription factor NusE. NusG was therefore proposed to play a prominent role and couple transcription and translation.

However, recent cryo-EM reconstructions of a 30S ribosomal subunit in complex with RNAP core and of a translating 70S ribosome in complex with a stalled RNAP (the latter complex was termed expressome) were incompatible with this model, because neither architecture would allow simultaneous binding of NusG-NTD to RNAP and NusG-CTD to uS10 [START_REF] Demo | Structure of RNA polymerase bound to ribosomal 30S subunit[END_REF][START_REF] Kohler | Architecture of a transcribing-translating expressome[END_REF]. To explore this phenomenon and to resolve some of these controversies, the host lab aimed to obtain reconstructions of a complex, which mimics a ribosome approaching RNAP. I was also involved in some stages of this project and will describe my contributions in the Results section.

VIII. OBJECTIVE

Structural studies help to gain a mechanistic understanding of fundamental biological processes. Recently, single particle cryoelectron microscopy (cryo-EM) has revolutionized the field of structural biology because it facilitates more rapid determination of highresolution structures of increasingly complex assemblies. The main reason for these advancements are technical breakthroughs such as: i) the development of direct electron detectors [START_REF] Faruqi | Electronic detectors for electron microscopy[END_REF]McMullan et al., 2016); ii) 300 keV microscopes with increased beam penetration, less inelastic scattering, and more coherent illumination such as the Titan KRIOS; iii) novel software developments to compensate for beam induced particle motion [START_REF] Zheng | MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy[END_REF]; and iv) improved software for data processing, and a posteriori correction of optical aberrations [START_REF] Punjani | CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination[END_REF][START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF].

Moreover, single particle cryo-EM allows us to observe: i) biological samples close to their native state; ii) different states of a complex within the same sample; and iii) large and increasingly complex assemblies containing proteins and nucleic acids.

RNAP is universally conserved in all three kingdoms of life. Likewise, it is believed the molecular mechanism of transcriptional pausing is also highly conserved from prokaryotes to mammals, according to recent structural studies [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]Kang et al., 2018b;[START_REF] Vos | Structure of paused transcription complex Pol II-DSIF-NELF[END_REF].

II. GENOMIC DNA EXTRACTION

1.5 mL of a saturated overnight E. coli culture was harvested by centrifugation for 1 min at 3724 g. The cell pellet was vortexed and lysed in 600 μL DNA lysis buffer (10 mM Tris acetate pH 8.0, 1 mM EDTA, 10% SDS, 20 μg Proteinase K), followed by incubation for 1 h at 37°C.

An equal volume of phenol/chloroform was added, and the tube was gently inverted until the phases were completely mixed. Following centrifugation for 5 min at 25,000 g at room temperature, the upper aqueous phase was transferred to another 1.5 mL tube. This phenol/chloroform extraction step was repeated until the white protein layer was no longer visible. The remaining phenol was then removed by adding an equal volume of chloroform to the aqueous layer.

After centrifugation for 5 min at 25,000 g, the extracted supernatant (aqueous phase) was transferred to a new tube. The DNA was precipitated by adding 2.5 to 3 volumes of cold 100% ethanol, 30 min of incubation at -20°C, and sedimentation at 25,000 g for 15 min at 4°C.

The DNA pellet was washed with 1 mL of 70% ethanol, followed by centrifugation for 2 min at 25,000 g. ii. NUSG NTD GENE Vector pIA_244_Eco_nusG (see above) was also used as a template to amplify the N-terminal region of E. coli NusG (NusG-NTD, M1-R123) by PCR using the Q5 DNA polymerase enzyme (New England Biolabs; PCR annealing at 55°C). Primer sequences are shown in table S1.

Oligonucleotide primers contained the restriction site NdeI and NotI.

The PCR product was digested and ligated into the pAX0_His10_HRV3C_Eco_nusG_NTD vector (protein expression vector).

IV. DNA/RNA OLIGONUCLEOTIDES DNA (TriLink) and RNA (Dharmacon) oligonucleotides were chemically synthesized and desalted by the manufacturer. RNA was deprotected following the protocols provided by the manufacturer. Both DNA and RNA were dissolved in RNase free water and aliquots were stored at -80°C.

V. PROTEIN PURIFICATIONS NusA was eluted using a gradient over 10 CVs into lysis buffer plus 1 M NaCl. The peak was concentrated and further purified by gel filtration using a Superdex 75 16/60 (GE Healthcare) column equilibrated with GF buffer (10 mM HEPES pH 8.0, 0.15 M NaCl, 0.1 mM EDTA, 1 mM DTT).

The final protein was concentrated to >50 mg/mL, aliquots were flash frozen, and stored at -80°C.

iii. PURIFICATION OF RNAP pVS11_rpoA_rpoB_rpoC_HRV3C_His10_rpoZ vector containing the genes encoding the E. coli RNAP core enzyme subunits with a C-terminal His10-tag on the β'-subunit was transformed into E. coli LACR II. Staining Solution, BIORAD). 10 mL of culture was harvested and kept for solubility test (3724 g, 30 min, 4°C).

ii. SOLUBILITY TEST

This solubility test protocol is a common procedure for all proteins.

Buffer A used to lyse the cells can be adjusted for each specific protein.

Pelleted cells from 10 mL culture were suspended in

X. IN VITRO TRANSCRIPTION ASSAYS

The DNA constructs containing different inserts involved in the different transcription assays were generated by restriction-ligation method.

i. RNA EXTENSION ASSAY

RNAP ECs were assembled as described in the previous section but ii.

OPS-PAUSE ASSAY

For the ops-pause assay, the template DNA contained a T7A1 promoter followed by a U-less region (A48) and the pause site (ops

iii. HIS-PAUSE ASSAY

To monitor the effect of NusG with or without NusA on the his-pause elongation complex (his-PEC) a previously published experimental setup was used [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]. The nucleic acid scaffold was prepared by annealing RNA and DNA as described for unlabelled nucleic acid scaffolds before (Reconstitution of nucleic acid scaffold (RNA:t-DNA:nt-DNA)). The nucleic acid scaffold (0.5 μM) was first mixed with RNAP (1 μM) in EM buffer supplemented with 0.02 mg/mL acetylated BSA at 37°C for 10 minutes. The 27nt-RNA in the complex was first extended and radioactively labelled by incorporation of 32P-α-rCTP (30 mCi) at 37°C for 1 minute, followed by addition of non-radioactive rCTP (2 μM final) and rUTP (100 μM final), extending the RNA to the position of the transcriptional pause at an RNA length of 29nt. The his-PEC was then incubated with transcription factors at 37°C for 2 minutes, in the following mixtures: 1) RNAP; 2) RNAP + NusA; 3) RNAP + NusG-FL; 4) RNAP + NusA + NusG-FL; 5) RNAP + NusA + NusG-NTD. To follow pauseescape over time, the next nucleotide rGTP (10 μM) was added at room temperature, samples were taken at regular time intervals (7'', 14'', 21'', 30'', 45'', 1', 1'30'', 2', 2'30'', 3', 5', 10', 15') and fullcon promoter DNA (1 μM). Samples were taken after indicated time intervals (10'', 20'', 30'', 1', 1' 30'', 2', 3', 5', 10') and the reactions were stopped by mixing with equal volume of stop buffer (8 M urea, 20 mM EDTA pH 8.0, 5 mM Tris-HCl pH 7.5, 0.5% bromphenol blue, and 0.5% xylene cyanol). At the end, rNTPs (5 mM) were added to drive all reactions to completion for 5 minutes. RNA products were separated and visualized as described above. Termination efficiencies were calculated as the ratios of the total terminated RNA products (or the terminated RNA products at a given termination site) over the sum of RNA that has reached the termination site or bypassed it.

v. RNA MARKER

To better identify the termination sites, I prepared a radioactively labelled DNA marker to estimate the size of the RNA species. Plasmid DNA for pBR322 (1 mg/mL) was pre-digested with restriction enzyme MspI (NEB). 5'-phosphates were removed by adding 20 U of calf intestinal phosphatase (NEB) and the vector fragments were purified by standard phenol/chloroform extraction and alcohol precipitation (see 'Genomic DNA extraction'). The resuspended fragments were labelled with 32P-γ-rATP using T4 polynucleotide kinase (NEB) as described in the section of 'labelling of RNA or DNA oligonucleotides' and stored at -20°C.

XI. SAMPLE PREPARATION

To prepare complexes for cryo-EM, the nt-DNA, t-DNA and RNA oligonucleotides were mixed in 2:2:1 molar ratio and annealed in a PCR machine (2 minutes at 95°C, 2 minutes at 75°C, 5 minutes at 45°C, slow cooling to 4°C at a rate of -2°C/minute, hold at 4°C) in reconstitution buffer (10 mM Tris-HCl pH 8.0, 40 mM KCl, 5 mM MgCl2). The RNAP-NusA-NusG complex was formed by mixing RNAP, nucleic acid scaffold, NusG and NusA in 1:2:3:3 molar ratio in EM buffer (10 mM HEPES pH 8.0, 100 mM KOAc, 1 mM DTT, 10 μM ZnCl2, 5 mM Mg(OAc)2, 150 μM EDTA, 1 mM DTT, 0.02 mg/mL acetylated BSA), incubated at 37°C for 5-10 minutes. For the other two complexes, RNAP-NusA and RNAP-NusG, the components were mixed with the same molar ratios. Each complex was purified by gel filtration (Superose 6 10/300 GL, GE Healthcare).

The complex was then concentrated to 7-10 mg/mL using Amicon Ultra centrifugal filter units with a 10kDa molecular weight cut-off. CHAPSO

XII. CRYO-EM DATA COLLECTION AND PROCESSING

Images were recorded using Serial EM on two different Titan Krios (FEI) microscopes at 300 keV of acceleration voltage and each equipped with a K2 Summit camera (Gatan, Inc., Pleasanton, CA, USA) placed at the end of a GIF Quantum energy filter (Gatan, Inc.) in zero-energy-loss mode with a slit width of 20 eV (IGBMC, Illkirch, France, Biozentrum Basel, Switzerland). The target defocus ranges were set to -0.8 to -3 μm.

The movies contained 40 frames, which were collected in superresolution counting mode with pixel size of 0.55 or 0.52 Å/pixel, and exposures ranging from 5 to 6.4 e-/A2/s, which corresponds to a total dose of ~ 50 e-/Å2.

The images were first motion-corrected and dose weighted using Motioncor2 (Mastronarde, 2005). The pixel size of micrographs was rescaled with relion_image_handler to 1.09 Å/pixel in order to combine datasets collected on different microscopes. The contrast transfer function (CTF) for each micrograph was then estimated using CTFFIND4 (Rohou & Grigorieff, 2015). The particle-picking templates were generated using semi-automated swarm method in EMAN2 [START_REF] Tang | EMAN2: An extensible image processing suite for electron microscopy[END_REF]. Automatic particle picking was done on the lowpass-filtered (20 Å) and contrast-inverted micrographs using Relion [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF].

The particles were extracted from the dose-weighted micrographs and the resulting datasets from the same complex were merged. Joined particles were re-extracted with 4x4 binning and further sorted by 2D classification. Selected particles were re-extracted with 1x1 binning and used for 3D refinement using an ab-initio model generated in cryoSPARC [START_REF] Punjani | CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination[END_REF] and 3D classification in cryoSPARC and in Relion. Further 3D refinements, heterogenous refinements and 3D variability analyses were performed in cryoSPARC. [START_REF] Emsley | Coot: Model-building tools for molecular graphics[END_REF]. Furthermore, the upstream and downstream DNA duplex were built de novo in Coot. The resulting models were real-space refined using secondary structure restraints and geometry optimization in Phenix against density maps sharpened by applying a B-factor that was estimated using automated procedures.

XIII. STRUCTURAL MODELLING

STRUCTURAL AND FUNCTIONAL STUDIES ON THE EFFECTS OF E. COLI NUSA AND NUSG ON TRANSCRIPTION REGULATION

The first result section is dedicated to the regulation of NusA and NusG on EC. for 3 hours. Overexpressed E. coli NusG-FL (20.5 kDa) and NTD (13.5 kDa) were confirmed by SDS-PAGE analysis (Figure 10). In parallel, the expression of proteins from T. aq or T. th were verified with the same method (Figure 11).

Despite good overexpression of all proteins, the purification was not successful for any of them. The majority of the protein remained in the insoluble fraction of the lysate, which was confirmed by comparing insoluble cell debris (pellet) and supernatant (SN) by SDS-PAGE (Figure 12).

II. BIOCHEMICAL FUNCTIONAL VALIDATION

Before proceeding to structural studies, I performed biochemical tests to see whether the proteins could form functional complexes in vitro.

i. I) ELECTROMOBILITY SHIFT ASSAY (EMSA)

First of all, I wanted to verify whether these separately purified proteins

b) T . T H N U S G -T. T H RNAP C O M P L E X

After obtaining RNAP-scaffold-NusG complex results for E. coli, I performed similar experiments with T. th NusG (Figure 17) to confirm its binding to T. th RNAP. I then tried to add NusA to the RNAP-scaffold-NusG complex. Figure 17 shows four complexes that were formed: RNAP-scaffold; RNAP-scaffold-NusG; RNAP-scaffold-NusG-NusA and RNAP-scaffold-NusA.

Figure 17 Gel shift assay for detection of T. th EC formation.

A) The gel was first stained with Coomassie brilliant blue. B) The gel was then stained with ethidium bromide. The different components in the samples are indicated on the top of the gel.

c) RNAP -S C A F F O L D C O M P L E X

To verify that each nucleic acid strand was present in the complex, I individually labelled them with radioactive isotope 32P (Figure 18). DNA was used in a 2-fold molar excess over RNA giving rise to two bands:

DNA alone or RNA bound to DNA. In presence of the T. th RNAP, a band at the top corresponds to the RNAP-scaffold complex. Individual oligonucleotides were labelled by 32P (marked in colour red, blue and green) and assembled into a complete scaffold. Addition of RNAP results in complex formation (RNAP + S) and a shift of the scaffold (S). Bottom: the same gel stained with ethidium bromide.

ii.

IN VITRO TRANSCRIPTION ASSAY

After confirmation of the complex formation with E. coli NusG or T. th NusG, I wanted to investigate the role of NusG in transcriptional pausing.

E. coli

NusG is known to increase the overall transcription rate and decrease pausing stabilized by RNAP backtracking, which is the most readily observable in vitro phenotype of E. coli NusG [START_REF] Burova | Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro[END_REF]. For that reason, I selected the E. coli ops pause (ops -operon polarity suppressor) to monitor NusG activity. The ops-pause is biochemically well characterized and is representative for a pause, stabilized by RNAP backtracking [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF].

The template for this assay contains a strong T7A1 promoter, followed by a 48-nt long U-less region (A48) and an ops-pause sequence (Figure 19 A). I cloned this template into pIA171 vector and amplified it by PCR.

By addition of σ 70 factor RNAP initiates the transcription from the promoter. In absence of rUTP, transcription proceeds until the end of A48, where the elongation complex halts. This enables radioactive labelling of RNA transcripts with 32P-α-rCTP and synchronisation of transcription (Figure 19 B). Upon addition of all four rNTPs, RNAP resumes transcription and pauses at the ops-site, which is observable as accumulation of RNA product at U60 that disappears over the time.

a) F U N C T I O N A L T E S T O N A S C A F F O L D

After I confirmed that purified NusG was functional, I continued the experiments with an RNA extension assay to ensure that a functional elongation complex forms and maintains its function on a minimal nucleic acid scaffold (see e.g. Figure 15). I performed the elongation reaction by monitoring one base pair extension. As expected, Figure 20 demonstrates that RNAP specifically incorporates rCTP at the active site.

Taking all together, my ECs with or without NusG were both functional.

Similar functional tests had verified that TF NusA is functional in the lab before. The next step will be structural studies. The peak fraction containing the reconstituted complex is then concentrated and used for grid freezing. The EM data is collected as described in the Materials and Methods section. Data processing, refinement, model building, and structural refinements were carried out using EMAN [START_REF] Tang | EMAN2: An extensible image processing suite for electron microscopy[END_REF], Relion [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF], cryoSPARC [START_REF] Punjani | CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination[END_REF]), Chimera (Pettersen Ef Fau -Goddard et al., 2004), Phenix (Adams et al., 2012), and Coot [START_REF] Emsley | Coot: Model-building tools for molecular graphics[END_REF]. (Kang et al., 2018a;[START_REF] Krupp | Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor[END_REF].

III. STRUCTURAL STUDIES

The overall structure of RNAP is not rigid. The regions with the highest flexibility are highlighted and labelled above: β-lobe (β143 -β448), SI2 (β938 -β1039) and SI3 (β945 -β1130).

Figure 23 Comparison between NusG-EC, NusG-opsEC and λN-TAC.

Figure 24 The flexible domains in RNAP.

RNAP is shown as a surface and NusG as a cartoon. RNAP is coloured in blue-gray, NusG in red, the nt-DNA and t-DNA in lavender and violet, respectively. The contact regions (coloured in cyan) are defined as the residues in RNAP that are closer than 5 Å to NusG. Image in the inset shows some of the hydrophobic interactions (yellow patches) between NusG and RNAP. The clamp and shelf modules of RNAP (together referred to as the swivel module) can oscillate between a non-swivelled conformation, which is believed to be a requirement for substrate binding by RNAP (Kang et al., 2017(Kang et al., , 2018a)), and a swivelled conformation, characteristic for paused RNAP [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]Kang et al., 2018b).

To further investigate the dynamics of NusG-EC, I performed heterogeneous refinements in CryoSPARC. Particles were initially sorted into five classes (Figure 27). Among these, the conformation of the RNAP swivel module in a class lacking NusG appears to be the least swivelled ('w/oG swi-'). Here the RNAP swivel module is in a similar conformation to previous reconstructions (Abdelkareem et al., 2019).

Therefore, I used this class as the reference for swivel module movements (Figure 28). The consensus structure of NusG-EC from all particles in the dataset refined to a state most similar to a non-swivelled conformation, hinting at the equilibrium of swivel module movements being close to a non-swiveled state in the NusG-EC (swivelling angle 1.4°) (Figure 29). Figure 29 The consensus structure of NusG-EC is most similar to the non-swivelled conformation.

Figure 28 Similarity between 'w/oG swi-' class and backtracked complex.

Two classes (40% of particles, 'w/oG swi-', and 'w/oG swi++') contained either very weak or no discernible density for NusG (Figure 27). These two classes thus represent an RNAP EC not bound to NusG but nevertheless differ in their swivel module conformation. The swivelling angle of RNAP in 'w/oG swi++' class is 2.4° compared to the reference structure ('w/oG swi-') (Figure 30). This indicates that RNAP can access Furthermore, in all classes with NusG, the C-terminal domain (CTD) of NusG and the flexible flap-tip helix (FTH) of RNAP only become visible in low-pass-filtered maps (Figure 34). NusG-CTD appears as extra density on top of the FTH. Together with NusG-NTD it arcs around the upstream DNA, which may limit its mobility and further stabilize it (Figure 35). A paralogue of NusG, RfaH, also binds the FTH with its CTD in a similar manner (Kang et al., 2018a). Previous studies suggested that NusG-NTD might be incompatible with the swivelled state due to steric clashes (Kang et al., 2018a). To find the clashes in the swivelled state, I used the NusA-his-PEC which was obtained in our lab previously [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]. NusA-his-PEC represents a paused RNAP stabilized by an RNA hairpin, which forms in the nascent transcript in the RNAP exit channel (his-RNA). The RNAP in this complex adopts a highly swivelled state, which should be incompatible with the presence of NusG. However, when I aligned NusG-EC with NusA-his-PEC [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF] based on the RNAP core module or swivel module, no major clashes could be seen because the tip of the β-protrusion is flexible and can move away from NusG during swivelling (Figure 36). Therefore, steric clashes do not appear to be the main reason for NusG to restrict RNAP swivelling. An alternative explanation is that NusG binding might favour a non-swivelled conformation of RNAP by forming additional contacts with the β-lobe (A380, Y367, G373) and the β-protrusion (Y62).

To summarize, the NusG-EC reconstructions showed that in absence of NusG, RNAP can undergo swivel movements thought to play an important role for transcriptional pausing. In presence of NusG, the NusG-NTD binds to the previously reported site on RNAP by hydrophobic interactions, and the CTD is able to interact with the RNAP FTH in a similar manner as RfaH-CTD. Contrary to previous assumptions, I observe that RNAP is still able to swivel in presence of NusG, albeit not to the extent observed for paused RNAP complexes. NusG might stabilize the upstream and nt-DNA to favour RNAP in a less swivelled conformation. ii.

RNAP-NUSA COMPLEX

The same nucleic acid scaffold (Figure 21 A) was used to obtain a reconstruction of an RNAP-EC bound to NusA (NusA-EC) with a nominal resolution of 4.1 Å (Figure 37). NusA refined to lower local resolution because of its intrinsic flexibility and rotation relative to RNAP, which is consistent with earlier observations (Figure 38) [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF][START_REF] Krupp | Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor[END_REF]. The overall structure resembles a NusA bound paused RNAP elongation complex at the E. coli his-pause (NusA-his-PEC, backbone RMSD for RNAP 1.3 Å, Figure 39), but it is less swivelled compared to the NusA-his-PEC (Figure 40). In both structures, NusA is positioned close to the RNA exit channel and binds to the β-flap tip helix (FTH), the C-terminal domains of the RNAP α-subunits (α-CTDs) and the C-terminal tip of the ω-subunit. However, in contrast to the paused state, RNAP is post-translocated with an unpaired template DNA base accommodated in the active site (Figure 37). Figure 39 Similarity between NusA-EC and NusA-his-PEC.

Figure 40 NusA-EC differs compared to NusA-his-PEC in the swivelling angle.

The swivel module of NusA-EC is less swivelled compared to NusA-his-PEC. NusA-EC is coloured in blue and NusA-his-PEC in grey.

In a consensus refinement of the NusA bound EC, the swivel module appears to be in an intermediate position (2.4°) compared to the reference structure ('w/oG swi-' class in non-swivelled state, Figure 41).

Heterogenous refinement in CryoSPARC [START_REF] Punjani | CryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination[END_REF] at first separated two classes: one (around 65% of particles) adopted a more swivelled but post-translocated state, while the other (remaining 35% of particles) were in a non-swivelled post-translocated state. An independent 3D classification in Relion [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF] gave similar result. To better model the conformational dynamics of the complex, I employed 3D variability analysis in CryoSPARC (the results

were confirmed with a round of heterogenous refinement followed by 3D refinement). The RNAP swivel module has access to a continuum of positions where the swivel module in the most extreme position is rotated by 5.5˚ relative to the non-swivelled state (Figure 41).

Interestingly, density for the RNA-DNA hybrid is generally weaker and less defined in swivelled conformations as opposed to the nonswivelled (Figure 42), which is consistent with the idea that nonswivelled RNAP is prone for catalysis. In general, the upstream DNA and nt-DNA are more disordered in the NusA-EC compared with NusG-EC (Figure 43). Both cryo-EM density maps are displayed at comparable contour levels. Protein and nucleic acids are shown as sticks. The EM density for the DNA-RNA hybrid is better defined in the nonswivelled state.

There is additional density visible between NusA and the RNA exit channel in the reconstruction of the swivelled conformation, which I interpret as corresponding to the nascent RNA. However, it cannot be modelled accurately due to high flexibility (Figure 44). Figure 44 shows that S1, an RNA binding domain of NusA, is positively charged and interacts with the putative RNA. Two other RNA binding domains follow the S1 domain in NusA (KH1 and KH2), consistent with my observations and with previous structural results and proposals that the RNA transcript is guided along NusA as it exits from RNAP [START_REF] Beuth | Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ[END_REF][START_REF] Said | Structural basis for λN-dependent processive transcription antitermination[END_REF]. The zinc finger (ZF) of RNAP is positively charged and seems to interact with nascent RNA. In absence of RNA, the RNAP ZF and S1 likely repel each other. In presence of nascent RNA, the RNA's negative charge could stabilize the RNAP ZF and S1 in a proximal position. Furthermore, RNAP also provides a positively charged path for the nascent RNA, guiding it to the RNA binding domains of NusA (Figure 44 A) [START_REF] Beuth | Structural basis for the interaction of Escherichia coli NusA with protein N of phage λ[END_REF][START_REF] Said | Structural basis for λN-dependent processive transcription antitermination[END_REF]. 3D variability analysis, as

well as heterogeneous refinement, showed that the interaction patterns between NusA, RNAP and the nascent RNA change during swivelling. The density of the single-stranded RNA near the RNAP ZF can only be observed in the swivelled state. In the non-swivelled state, the single-stranded RNA density disappeared, and the ZF moves further away from NusA (Figure 44). When RNAP transits to the non-swivelled state, the C-terminal tip of the omega subunit moves closer to NusA and appears to establish a contact instead.

To conclude, NusA appears to favour higher swivelling angles than seen in the NusG-EC. This is reflected in an increased swivel angle seen in the consensus refinement (the equilibrium of the ensemble). to model the pause-escape rates similar to a previous study [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]. Please note, that this equation contains two exponential terms to account for two kinetic species of RNAP: RNAPfast and RNAPslow, which represent 'fast' and 'slow' pause-escape rates, respectively. One ii.

OPS-PAUSE ASSAY

Next, I wanted to test combinatorial effects of NusA and NusG on the ops-pause, to see if the two factors are cancelling their effect similar to my observations at the his-pause. I used the same template DNA (pIA171_ops) as for the NusG functional assay (Figure 50 A). The addition of TFs does not affect the position of the pause site.

Furthermore, I quantified the RNA species at the ops-pause site to estimate the pausing effect in presence of different TFs. I fit the data using a first order rate law to model the pause escape rates. However, I found that a single exponential (representing one single species in the molecular ensemble) was not always sufficient to model the experimental results. This suggested the existence of two kinetic species in the molecular ensemble. Therefore, the two species were defined as 'fast' (RNAPfast) and 'slow' (RNAPslow). However, I would like to emphasize that this assumption is based on a small number of experimental measurement points and thus error prone.

About a third of RNAP alone (37%) enters the pause at the ops-site 24% in its absence), while the fraction of RNAPslow is similar (17% vs. 13%). Second, NusA affects the pause escape rates of both species and reduces them by a factor of 2 to 5 (0.08 sec-1 vs. 0.13 sec-1, and 0.004 sec-1 vs. 0.02 sec-1). As a result, the overall half-life of RNAP on the opspause is much longer in presence of NusA. As a conclusion, NusA has two effects: it appears to increase pause probability and pause duration.

(
However, the common belief was that NusA has no effect on the backtracked pause [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF], which does not agree with my observation. Remarkably, in presence of both NusA and NusG, the individual effects cancel each other and RNAP behaves similar to a situation where both factors are absent (Figure 50 C-E, compare green and grey curve, compare escape rates and fraction of RNAPfast, and RNAPslow). studies have demonstrated that NusG-CTD stimulates ρ to adopt the closed-ring state [START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF]. It has also been observed before that in presence of NusG, termination occurs earlier [START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF]. This phenomenon was termed as 'early termination'. More recently, it has also been observed that ρ can directly bind and inactivate the RNAP EC in a translocation independent manner (Said et al., Science 2020, Hao et al., Mol Cell 2020). This result is consistent with earlier biochemical data, which suggested that ρ interacts with NusA and can bind RNAP independent of RNA (Schmidt et al., JBC 1984, Epshtein et al., Nature 2010). Interestingly when both NusA and NusG were presented in the ρ-EC, no direct interaction between the NusG-CTD and ρ has been observed (Said et al., Science 2020). However, neither study excluded the role of NusG on ρ-dependent-termination, crosslinking indicated that NusG-CTD might transiently bind ρ, and NusG-CTD may for example play a role later in the termination pathway (Hao et al., Mol Cell 2020).

My structural studies suggest once NusA occupies the FTH, the CTD of NusG is in principle free to potentially interact with ρ. To determine termination efficiency in presence of combinations of these factors, I cloned a template DNA containing a rut termination sequence (Figure 51) that is frequently used in termination assays [START_REF] Faus | Sequences in the 5' proximal segment of the paused transcript affect NusA-mediated enhancement of transcriptional pausing[END_REF]. The transcription assay was performed similarly as for the ops-pause assay described above. RNAP can either release the transcript at termination sites or by-pass all terminators and fall off at the end of the linear template DNA (run-off). The terminated products on the gel at the early and late termination sites are determined with the help of a DNA ladder (see the 'RNA marker' section in the 'Materials and Methods' part). The cumulative termination (Figure 52) showed that ρ factor improved termination efficiency (the fraction of total RNA, which terminates within a given termination region) up to 45% compared to RNAP in absence of any TFs, which exhibited a termination efficiency of only 13%. Addition of NusA reduced the termination efficiency to 23%, but addition of NusG enhanced this effect to 51% (Burns et al. reported a 3fold increase, [START_REF] Burns | Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli[END_REF]). When both factors were present in the reaction, the termination efficiency increased further and reached 56%. RNAP+ρ+NusA+NusG-NTD). The results showed that NusG-NTD is no longer stimulating early termination by ρ and the transcription profiles resembles a reaction using RNAP, ρ, and NusA but lacking NusG altogether (Figure 54 A). Thus, the NusG-CTD is essential for its stimulatory activity in ρ-dependent termination.

The late termination region consists of several distinct termination sites, which might be independent from each other, and therefore cannot be described with a simple equation as the one used for the early termination region.

THE ROLE OF E. COLI NUSG IN THE EXPRESSOME COMPLEX

Evidence accumulated since the mid 1960s suggesting that transcription and translation are physically and possibly also kinetically coupled in most prokaryotes [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF][START_REF] Byrne | THE IN VITRO FORMATION OF A DNA-RIBOSOME COMPLEX[END_REF][START_REF] Feklistov | Structural basis for promoter -10 element recognition by the bacterial RNA polymerase σ subunit[END_REF][START_REF] Johnson | Structural basis of transcription arrest by coliphage HK022 nun in an escherichia coli rna polymerase elongation complex[END_REF][START_REF] Miller | Visualization of bacterial genes in action[END_REF][START_REF] Plaschka | Cooperation between translating ribosomes and RNA polymerase in transcription elongation[END_REF][START_REF] Saxena | Escherichia coli transcription factor NusG binds to 70S ribosomes[END_REF][START_REF] Zhu | Disruption of transcription-translation coordination in Escherichia coli leads to premature transcriptional termination[END_REF]. Previous studies, which I described in the introduction on the NusG-CTD, indicated that NusG-CTD interacts not only with ρ termination factor but also with the ribosome in vivo and in vitro [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF][START_REF] Saxena | Escherichia coli transcription factor NusG binds to 70S ribosomes[END_REF] and thus may form a physical bridge. A direct interaction between NusG and the ribosome has been proposed as a strategy to prevent premature termination [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF][START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF] because it would prevent NusG to interact with ρ. However, this model has been challenged because a recent low-resolution reconstruction of the E. coli RNAP coupled to the E. coli 70S ribosome was not compatible with NusG forming a physical bridge [START_REF] Kohler | Architecture of a transcribing-translating expressome[END_REF]. It is important to know if and how NusG connects the expressome complex (ribosome in complex with a RNAP) to better understand the role of NusG with different partners. The final part of my results section describes my involvement in a project that aimed to elucidate the role of NusG in the coupling of transcription and translation.

This project was started by Maria Takacs in the team. She purified most of the components and assembled a complex of the 70S ribosomes, tRNAs, RNAP, NusG, and a DNA-mRNA scaffold. I purified NusG and was involved at the beginning of the project during sample optimization. I helped with sample preparation, collecting single-particle datasets on the Polara, as well as collecting data on the Titan KRIOS. I helped Maria with data processing and with the analyses of the first reconstructions.

Indeed, we encountered many problems. For instance, the prevalence of the expressome complex is low in comparison to free ribosomes. This implies more extensive data processing (removal of free ribosomes by 2D and 3D classification) and requires much larger data sets to compensate (only about 10% to 20% of particles were expressomes).

We also had to overcome problems with preferential orientation, which we tried to compensate for by using carbon coated grids. Surprisingly, we found more than one RNAP binding site on the ribosome (Figure 55).

Both had been observed before individually [START_REF] Demo | Structure of RNA polymerase bound to ribosomal 30S subunit[END_REF][START_REF] Kohler | Architecture of a transcribing-translating expressome[END_REF]. Even though RNAP is a large 400kDa-enzyme, particle alignment is dominated by the ribosome and the EM density of RNAP is fragmented due to its flexibility relative to the ribosome. All these problems described above increased the complexity to obtain highresolution reconstructions of these expressome complexes.

Michael Webster joined the team as a postdoctoral researcher and contributed to this project. Using mRNAs of different lengths, a series of structures representing uncoupled, coupled, and collided expressome states were determined by cryo-electron microscopy [START_REF] Webster | Structural basis of transcription-translation coupling and collision in bacteria[END_REF]. Focussed refinement approaches allowed Michael and Maria to obtain high resolution reconstructions and build atomic models. Without NusG but with sufficient distance of shared mRNA between RNAP and the ribosome, the mRNA is the only consistent connection and the resulting assembly is highly flexible (RNAP can almost freely rotate around the mRNA axis and translate by more than 50 Å). Analysis of RNAP orientations relative to the ribosome suggested seven clusters of preferred orientations. Addition of NusG results in the formation of a physical bridge. This coupled expressome state restrains RNAP into one cluster relative to the ribosome, and aligns the mRNA transcript with the ribosomal helicase. Alignment of the nascent mRNA with the helicase likely prevents the formation of inhibitory secondary structures. Presumably this ensures smooth elongation for both enzymes and may represent an important functional role for coupling. Furthermore, in the NusG coupled state the NusG-CTD is not available to bind termination factor ρ. This provides therefore a structural explanation, how coupling prevents premature transcription termination by masking the interface of NusG-CTD, which may recruit and activate ρ [START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF].

When the ribosome approaches RNAP further along the shared mRNA, the components adopt an orientation matching a previously reported low-resolution reconstruction of the expressome [START_REF] Kohler | Architecture of a transcribing-translating expressome[END_REF] and we concluded it likely represents a collided state, where translation has halted.

Maria and Michael also confirmed the two RNAP binding sites on the ribosome. One site positions RNAP at the mRNA entrance channel of the ribosome and is consistent with coupled transcription and translation. Occupancy of this site is mRNA dependent. However, if the mRNA does not support concurrent ribosome and RNAP binding, the site cannot be occupied by RNAP. The second site overlaps with ribosomal protein bS1 and is only occupied if bS1 is sub-stoichiometric.

The second site may play a role during translation initiation but its biological role remains unknown for the moment.

Figure 55 Early reconstruction of an expressome complex.

The initial map refined to reasonable high resolution for the ribosome but showed additional fuzzy density consistent with two RNAP binding sites (map on the left). The binding site labelled as position 2 (RNAP shown in blue) is consistent with a recent low-resolution reconstruction of RNAP bound to the 30S subunit (blue, position 2) [START_REF] Demo | Structure of RNA polymerase bound to ribosomal 30S subunit[END_REF]. Position 1 (RNAP in green) is consistent with a complex, which was formed by a translating 70S ribosome colliding with a stalled RNAP [START_REF] Kohler | Architecture of a transcribing-translating expressome[END_REF]. The RNAP in position 2 overlaps with ribosomal protein bS1 and saturation of ribosomes with bS1 appears to abolish RNAP binding in that position.

NusA and NusG -two TFs with opposing roles -have been studied for more than 40 years. However, until now it has remained unknown how they work together to affect transcription regulation. We know that RNAP adopts a swivelled conformation when it enters a pause.

However, we do not know whether RNAP switches between the swivelled and the non-swivelled conformations randomly or it swivels at pauses and un-swivels for elongation. In my PhD project, I demonstrate the dynamic of RNAP and the influence of two TFs on the elongation and termination of transcription.

I. THE SWIVELLED AND NON-SWIVELLED RNAP

During the elongation phase of transcription, when RNAP encounters a pause signal, it enters into an elemental pause. The elemental pauses appear very frequently throughout the genome, but additional factors are usually required to convert them into long-lived pauses that play key regulatory roles [START_REF] Larson | A pause sequence enriched at translation start sites drives transcription dynamics in vivo[END_REF][START_REF] Saba | Mechanism of bacterial transcription initiation: RNA polymerase -Promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis[END_REF][START_REF] Vvedenskaya | Interactions between RNA polymerase and the "core recognition element" counteract pausing[END_REF]. Recent studies illustrated that RNAP adopts a swivelled conformation during pausing [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]Kang et al., 2018b). It was suggested that the non-swivelled conformation is required for RNAP to be active and fold the catalytically important trigger helices to perform catalysis (Kang et al., 2017(Kang et al., , 2018a)). Furthermore, it is also consistent with my results where the RNA-DNA hybrid density map is more defined and more ordered in the non-swivelled state compared to the swivelled conformation. In other words, the non-swivelled conformation appears to increase the stability of the RNA-DNA hybrid and this likely contributes to a more effective catalysis during the transcription process. Surprisingly, I observed the swivelling movement of RNAP in all of my three RNAP complexes on a nucleic acid scaffold without a pause sequence (with NusA, with NusG, with both NusA and NusG, and even in a subset of particles not containing any TF). This suggests that swivelling might be an intrinsic and natural movement of

II. NUSA CONTRIBUTES TO PAUSING

NusA is known to be highly flexible and this is also the case when binding to an RNAP EC. It has been suggested that its flexibility is important for its regulation [START_REF] Said | Structural basis for λN-dependent processive transcription antitermination[END_REF]. Previous studies showed that NusA enhances pausing [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF][START_REF] Burns | Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli[END_REF][START_REF] Yakhnin | NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro[END_REF] and I suggest that it does so by Furthermore, the interaction between NusA and omega is disrupted when RNA was captured by ρ at priming state of the ρ-NusA-NusG-RNAP complex [START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. Such priming state shows a more swivelled conformation than the previous state, according to Said et al.

Taken together, NusA might be able to shift the equilibrium towards more swivelled states of RNAP and thus favour pausing and any other process that requires swivelling such as termination.

III. HALF-TRANSLOCATED STATE IS DEPENDENT ON THE DNA SEQUENCE

Two previous high resolution paused EC structures with or without

NusA showed that the RNA-DNA hybrid is in a half-translocated state (RNA 3'-end post-translocated, template DNA at intermediate position between pre-and post-translocated state) [START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF]Kang et al., 2018b). In addition, RNAP was observed to be in the swivelled

IV. NUSG CONTRIBUTES TO INCREASE THE ELONGATION RATE

NusG increases overall elongation rate. However, it has been shown that NusG does not increase the nucleotide incorporation rate nor does it increase the translocation rate of a non-paused RNAP [START_REF] Turtola | NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble[END_REF]. Thus it was concluded that NusG increases the elongation rate by suppressing pausing (Kang et al., 2018a). Recent studies showed that NusG prevents backtracked pausing and favours forward translocation by stabilizing the -10 bp in the DNA (Kang et al., 2018a;[START_REF] Turtola | NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble[END_REF]. Unfortunately, the map quality in my reconstruction as well as in the one from Kang et al. (Kang et al., 2018a) do not allow us to confirm this at the structural level. translocation as suggested previously (Kang et al., 2018a;[START_REF] Turtola | NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble[END_REF]. A similar strategy has been proposed for phage 𝝀 protein N, which stabilises the upstream DNA using a positively charged domain [START_REF] Krupp | Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor[END_REF].

V. NUSA AND NUSG COMPETE WITH EACH OTHER DURING ELONGATION

The two TFs NusG and NusA, which have opposing roles in E. coli, compete with each other at both the structural and biochemical levels.

The first supporting evidence is that both NusG-CTD and NusA-NTD interact with the FTH. 3D classification of the NusA-NusG-RNAP complex indicated the co-existence of complexes with both TFs bound to the RNAP EC but also complexes with individual TFs bound to RNAP.

The structure of RNAP is very dynamic, and it is influenced by both NusA and NusG. Their presence seems to affect the swivelling of RNAP:

according to the maximally swivelled structure, the swivelling angle in the NusA-NusG-RNAP complex is in between the one for the NusA-RNAP complex (more swivelled) and the NusG-RNAP complex (less swivelled). This indicates compensatory effects of these factors on the conformational equilibrium of RNAP. Moreover, NusA and NusG compete on different levels: 1) NusA binds RNA together with the RNAP ZF to favour RNAP in the swivelled conformation; 2) at very high swivelling angles NusG loses some of its interactions with RNAP and would therefore stabilize the complex in the 'non-swivelled' conformation. However, it does not prevent the swivelling movement by itself. This notion is not in full agreement with a previously published study (Kang et al., 2018a). When RNAP swivels, the protrusion module of RNAP moves away from the β-lobe to prevent clashes between NusG and RNAP. In addition, NusG gains more contacts with β-clamp to keep interacting with RNAP.

This apparent competition has also been shown at the biochemical level.

NusA is able to increase the fraction of paused RNAP species as well as the duration of hisand ops-pause. NusG has a more complex role during pausing. NusG can act on the pause-escape rate, it can also unify Different kinetic assays showed that both pause duration and pause frequency of RNAP can be modulated.

VI. NUSA & NUSG CONTRIBUTE TO TERMINATION

A process called termination needs to occur to end transcription. When RNAP arrives at the terminator, it first needs to pause in order to be able to terminate [START_REF] Gusarov | The mechanism of intrinsic transcription termination[END_REF] with or without the aid of TF. One of the TFs, the ρ factor can generate ρ-dependent termination. It has been proposed that the 'closed-ring' conformation of ρ is required for translocation along the nascent RNA and further trigger the dissociation of EC, thus termination [START_REF] Ray-Soni | Mechanisms of Bacterial Transcription Termination: All Good Things Must End[END_REF][START_REF] Richardson | Rho-dependent termination and ATPases in transcript termination[END_REF]. My results show that using 𝝀 tR1 ρ-dependent terminator, RNAP alone is not sufficient for generating termination and most RNAP bypasses it. ρ increases termination but does not increase the pause frequency or the pause duration at the terminator. In addition, previous studies show that NusA helps the ρ binding [START_REF] Epshtein | An allosteric mechanism of Rho-dependent transcription termination[END_REF][START_REF] Schmidt | Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein[END_REF]. Furthermore, in my study the addition of NusA increases the pause frequency and pause duration at the terminator, but it locates termination to regions further downstream can be interpreted as bypassed species from core terminator site. This can be supported by recent observation that NusA and SI2 of RNAP stabilized the ρ in the 'open-ring' conformation [START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. On the other hand, the TF NusG decreases the pause efficiency but enhances termination at the early terminator region. This correlates with previous studies [START_REF] Burns | Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli[END_REF][START_REF] Burns | Activation of Rhodependent transcription termination by NusG: Dependence on terminator location and acceleration of RNA release[END_REF][START_REF] Chalissery | Interaction surface of the transcription terminator rho required to form a complex with the C-terminal domain of the antiterminator NusG[END_REF][START_REF] Hao | Pre-termination Transcription Complex: Structure and Function[END_REF][START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF][START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. In the presence of both NusA and NusG, a cumulative and cooperative effect on increment of 'early' termination efficiency. The mechanism might be illustrated as following: NusA increases pausing efficiency at the early terminator providing time for NusG -ρ interaction. NusG-CTD is able to stimulate the 'closed-ring' conformation thus paused RNAP can be terminated. More recently, the role of NusG-CTD on ρ has been challenged because a series of single particle cryo-EM reconstructions showed that although in presence of both NusA and NusG, no direct interaction between the NusG-CTD and ρ has been observed [START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. Indeed, ρ can bind and inactivate the RNAP EC independently of translocation along RNA [START_REF] Hao | Pre-termination Transcription Complex: Structure and Function[END_REF][START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. However, neither study excluded a role of NusG-CTD and NusG-CTD-ρ cross-linking interactions for termination. In addition, my kinetic transcriptional assay indicated that NusG-NTD abolished the role of NusG, which also has been observed in several studies [START_REF] Chalissery | Interaction surface of the transcription terminator rho required to form a complex with the C-terminal domain of the antiterminator NusG[END_REF][START_REF] Lawson | Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein[END_REF]Mooney et al., 2009a;[START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. I suggest that NusG may interact and stimulate ρ in the steps afterward of these series of single particle cryo-EM reconstruction observation. To Likewise, we do not know the precise role of NusA in the expressome complex? For example, can E. coli NusA alone physically couple RNAP to the ribosome as observed for Mycobacterium pneumoniae? If so, does it have similar functional implications as NusG has on a coupled expressome?

Single particle cryo-EM has now reached a very mature state and allows us to study increasingly complex and very dynamic assemblies. Likewise, cryo-electron tomography (cryo-ET) can now achieve molecular resolutions. In the future we will continue to use single particle cryo-EM to obtain high resolution reconstructions to study our complexes of interest and use a reductionist approach but I believe we will combine it with cryo-ET and study target complexes also directly in situ. In my next step in my career, I would like to continue to use these methods to address fundamental biological questions and contribute to the science knowledge. The table shows some of the important structural modules of E. coli.

V. STRUCTURAL MODULES

FIGURE 1 :

 1 FIGURE 1: CRYSTAL STRUCTURE OF THE THERMUS THERMOPHILUS RNAP. ........................................................................ FIGURE 2 BACTERIAL PROMOTER DNA AND ITS INTERACTION WITH RNAP AND Σ FACTOR. .................................................... FIGURE 3 : THE 'DNA-SCRUNCHING' MODEL PROPOSED FOR TRANSCRIPTION INITIATION. ..................................................... FIGURE 4 : STRUCTURE OF ELONGATION COMPLEX WITH ITS ACTIVE SITE. ........................................................................... FIGURE 5 : NUCLEOTIDE ADDITION DURING ELONGATION............................................................................................... FIGURE 6 : STRUCTURE OF HEXAMER Ρ FACTOR. .......................................................................................................... FIGURE 7 : THE COMPARISON OF TWO TERMINATION MODELS. ....................................................................................... FIGURE 8 THE CONSENSUS SEQUENCE OF TRANSCRIPTIONAL PAUSING. .............................................................................. FIGURE 9 SCHEMATIC OF CLONED NUSG CONSTRUCTS. ................................................................................................. FIGURE 10 E. COLI NUSG EXPRESSION ANALYSIS. ........................................................................................................ FIGURE 11 EXPRESSION TEST OF T. TH OR T. AQ NUSG-FL OR NTD IN E. COLI LACR II. ....................................................... FIGURE 12 SOLUBILITY ANALYSIS OF E. COLI NUSG-FL ON 15%SDS-PAGE GEL. ................................................................ FIGURE 13 PURIFICATION OF NUSG. ........................................................................................................................ FIGURE 14 PURIFIED NUSA (A) AND RNAP (B) REVEALED BY SDS-PAGE AND STAINED WITH COOMASSIE BRILLIANT BLUE .......... FIGURE 15 SCHEMATIC OF THE DNA/RNA SCAFFOLD. ................................................................................................. FIGURE 16 GEL SHIFT ASSAY FOR DETECTION OF E. COLI RNAP-SCAFFOLD-NUSG COMPLEX. .................................................. FIGURE 17 GEL SHIFT ASSAY FOR DETECTION OF T. TH EC FORMATION. ............................................................................. FIGURE 18 VERIFICATION OF THE SCAFFOLD BINDING TO RNAP BY RADIOACTIVE LABELLING ON NATIVE 2% AGAROSE GEL. ............ FIGURE 19 TRANSCRIPTION ASSAY IN PRESENCE OF NUSG. ............................................................................................ FIGURE 20 ONE BASE PAIR EXTENSION ASSAY ON A SCAFFOLD. ........................................................................................ FIGURE 21 RECONSTITUTION OF HOMOGENEOUS ECS ON A CANONICAL POST-TRANSLOCATED SCAFFOLD. ................................. FIGURE 22 OVERVIEW OF CONSENSUS REFINEMENT OF NUSG-EC. .................................................................................. FIGURE 23 COMPARISON BETWEEN NUSG-EC, NUSG-OPSEC AND ΛN-TAC. .................................................................... FIGURE 24 THE FLEXIBLE DOMAINS IN RNAP. ............................................................................................................ FIGURE 25 CONTACT POINTS BETWEEN NUSG AND RNAP. ......................................................................................... FIGURE 26 THE DISTANCE BETWEEN THE SI3 (Β'945 -Β'1130) DOMAIN AND THE Β-LOBE. ................................................. FIGURE 27 HETEROGENOUS REFINEMENTS OF NUSG-EC IN CRYOSPARC. ...................................................................... FIGURE 28 SIMILARITY BETWEEN 'W/OG SWI-' CLASS AND BACKTRACKED COMPLEX. .......................................................... FIGURE 29 THE CONSENSUS STRUCTURE OF NUSG-EC IS MOST SIMILAR TO THE NON-SWIVELLED CONFORMATION. ................... FIGURE 30 CONFORMATIONAL CHANGE IN CLASSES LACKING NUSG. .............................................................................. FIGURE 31 SUPERPOSITION OF NUSG-EC CLASSES THAT CONTAIN NUSG. ....................................................................... FIGURE 32 COMPARISON OF NT-DNA AND UPSTREAM DNA DENSITY BETWEEN CLASSES WITH OR WITHOUT NUSG. .................. FIGURE 33 ELECTROSTATIC POTENTIAL OF NUSG-EC SURFACE. ..................................................................................... FIGURE 34 THE FLEXIBLE CTD DOMAIN OF NUSG...................................................................................................... FIGURE 35 THE UPSTREAM DNA IS ENCLOSED BY NUSG AND RNAP.............................................................................. FIGURE 36 Β-PROTRUSION OF RNAP MOVES AWAY WHEN NUSG BINDS TO THE EC. .......................................................... FIGURE 37 OVERVIEW OF CONSENSUS REFINEMENT OF THE NUSA-EC COMPLEX. .............................................................. FIGURE 38 STRUCTURAL HETEROGENEITY OF NUSA. ..................................................................................................

FIGURE 46

 46 FIGURE 46 HETEROGENEOUS REFINEMENT OF NUSA-NUSG-EC IN CRYOSPARC. .............................................................

L

  figure numéro 23). Les différences entre les trois structures sont majoritairement présentes dans les domaines mobiles de l'ARNP (SI3, b 945 -1130 ; SI2, b 937 -1040 ; et les éléments d'insertions spécifiques de lignée SI1, b 225 -345) (Figure 24). Dans les trois complexes, NusG-NTD interagit à la même position avec 'clamp hélices' b' clamp (264 -307) de l'ARNP, il entre en contact avec le squelette phosphate-sucre de l'ADN en amont de la bulle de transcription par l'intermédiaire d'une surface chargée positivement. Il semble également entrer en contact avec le domaine b protrusion (31 -139 et 456 -512) de l'ARNP et son lobe b (143 -448) (Figure 25). La distance entre le domaine SI3 et le lobe b varie parmi ces complexes, mettant en évidence la mobilité de ces domaines (Figure 26).

à

  NusG de guider et restreindre le mouvement de l'ADN. Cette intéraction pourrait favoriser la translocation vers l'avant et empêcher le retour en arrière de la polymérase. Une proposition similaire a été faite pour la protéine N du phage 𝝀 stabilisant l'ADN avec son domaine chargé positivement et favorisant ainsi la processivité de la ARNP. Des études antérieures ont suggéré que NusG-NTD pourrait être incompatible avec un état pivoté, en raison d'encombrement stériques. Cependant, selon nos reconstructions, le module pivotant peut pivoter jusqu'à 3,3 degré en présence de NusG car l'extrémité de la b-protrusion de l'ARNP s'éloigne de NusG lors du pivotement (Figure 36). Bien qu'ils ne soient pas incompatibles, les contacts supplémentaires avec le lobe (R371, Y367, A380) et la protubérance (Y62, S480) peuvent défavoriser un pivotement plus important. Dans les cartes filtrées en passe-bas à 10 Å, NusG-CTD et un élément de l'ARNP (FTH, Flap-Tip-Helix) deviennent visibles dans toutes les classes contenant NusG (Figure 34). NusG-CTD apparaît comme une densité supplémentaire au-dessus de la FTH. Un paralogue de NusG, le facteur de transcription RfaH, se lie également au FTH par son domaine C-terminal d'une manière similaire. Les domaines NTD et CTD de NusG connecte la sousunité b de l'ARNP, ceci permet d'enlacer l'ADN situé en amont de la bulle de transcription (Figure 35). Cette interaction pourrait aussi limiter la mobilité de l'ADN et ainsi le stabiliser. ii. NUSA-EC Nous allons maintenant comparer le résultat précédent avec le complexe de NusA-ARNP. Pour ce faire, j'ai reconstitué un complexe d'élongation contenant l'ARNP et les mêmes séquences d'acides nucléiques liée au FT NusA (NusA-EC). J'ai réussi à obtenir une structure à une résolution nominale de 4,1 Å (Figure 37). La densité correspondante à NusA dans les cartes obtenues, ne sont pas très bien résolues en raison de sa flexibilité intrinsèque et de son mouvement par rapport à l'ARNP (Figure 38), ce qui est cohérent avec les données présente dans la littérature (12). La reconstruction consensuelle ressemble à une ARNP en pause liée à NusA (RMSD du squelette pour la ARNP 1,7 Å, Figure 39) (12). NusA est positionné près du canal de sortie de l'ARN. Il se lie au FTH, aux domaines C-terminaux des sous-unités a (a-CTD) de l'ARNP et à l'extrémité C-terminale de la sous-unité w de l'ARNP. Dans la structure consensus de NusA-EC, le module pivotant apparaît dans une position intermédiaire (2,6 degré de rotations par rapport à la référence non-pivotante : classe sans NusG, Figure 41), ceci implique que cette structure est un mélange de conformations du module pivotant. Les classifications 3D dans CryoSPARC nous ont permis de séparer ces conformations en deux classes. La première contenant deux tiers des particules, qui a adopté un état pivoté post-transloqué. La deuxième contenant un tiers des particules, qui a adopté un état non-pivoté posttransloqué. Des résultats similaires ont été obtenus indépendamment en utilisant la classification 3D dans Relion. Pour mieux modéliser la dynamique conformationnelle, j'ai utilisé l'analyse de variabilité 3D dans CryoSPARC. Le module de pivotement de l'ARNP adopte un continuum de positions, le pivotement le plus important est de 5,5˚ par rapport à l'état non pivoté (Figure 41). NusA et l'ARNP forment un canal chargé positivement pour l'ARN naissant en le guidant vers les domaines de liaison de l'ARN de NusA (S1, KH1 et KH2). L'analyse de la variabilité 3D nous a également permis d'estimer la liberté conformationnelle de NusA et de l'ARNP pendant le pivotement. Parmi toutes les interactions de NusA avec l'ARNP, il semble qu'il y a une interaction dynamique entre eux. J'ai observé que le domaine S1 de NusA s'approche du doigt de zinc (ZF) de l'ARNP à l'état pivoté (Figure 44), alors que les domaines KH1 et KH2 de NusA s'approchent de la sous-unité w de l'ARNP à l'état non-pivoté. Nous proposons que NusA favorise l'état pivoté. En effet, le domaine S1 de NusA et ZF de l'ARNP sont tous chargés positivement (Figure 44), et ils se rapprochent en présence de l'ARN, ce qui permet indirectement de stabiliser le complexe dans l'état pivoté. De plus, la structure de NusA-EC (5.5 degrés) possède un mouvement plus important du module de pivotement par rapport à NusG-EC (3.3 degrés) ou à l'ARNP dépourvue de FT liés (2.4 degrés) (Figure 41 vs Figure 30, Figure 31). Ainsi, NusA favorise l'état pivoté, ce qui est corrélé avec son habilite de réguler la pause de l'ARNP. Le mouvement de pivotement de l'ARNP n'est donc pas exclusif aux complexes en pause, mais peut également se produire sans que l'ARNP sois dans son état de pause. Pourtant, il est intéressant de noter que la densité de l'hybride ARN-ADN semble plus forte et mieux définie dans la conformation non pivotée que dans la conformation pivotée (Figure 42), ce qui est accord avec la proposition selon laquelle l'ARNP non pivotée est plus proche de l'état actif. iii. NUSA-NUSG-EC Malgré leurs rôles opposés dans la transcription, NusA et NusG se lient simultanément à l'ARNP. Pour étudier cela, j'ai reconstitué un complexe d'élongation contenant l'ARNP, les mêmes séquences d'acides nucléiques utilisées précédemment et les FT NusA et NusG (NusA-NusG-EC). J'ai réussi à obtenir une structure à une résolution moyenne de 3,9 Å (Figure 45). La classification 3D a révélé deux classes principales : la première qui contient les deux facteurs liés à l'ARNP (65 % des particules, à une résolution 3,9 Å), tandis que la seconde correspond à NusG-EC (35 % des particules, à une résolution 4,1 Å) (Figure 46). Dans NusA-NusG-EC, les deux facteurs se lient sur leurs sites attendus de l'ARNP, en accord avec les deux structures précédemment présentées où chacun des FT est lié de manière individuelle avec l'ARNP. Même après filtrage passe-bas des cartes, aucune densité correspondante à NusG-CTD a pu être observée, ce qui suggère qu'il est très flexible. NusA et NusG semblent être en compétition pour lier le FTH de l'ARNP. Une classification 3D plus poussée a révélé l'ARNP pivote jusqu'à l'angle de 4 degrés (45 % des particules de la NusA-NusG-EC), mais la densité de NusG-NTD est plus faible dans cette sous-population, ce qui suggère une dissociation partielle (Figure 48). En comparant les structures de NusG-EC, NusA-EC avec NusA-NusG-EC nous remarquons, qu'en présence de NusA et NusG, l'amplitude de pivotement de l'ARNP semble se situer à un niveau intermédiaire (1,6 degrés rotation par rapport à la référence non pivotée : classe sans NusG, Figure 47), ce qui suggère que les deux facteurs compensent leurs effets respectifs. D'autre part, la liaison simultanée des deux facteurs peut également donner lieu à des effets coopératifs pour la mise en place de la terminaison de la transcription. iv. EFFETS DE NUSA ET NUSG SUR LA PAUSE HIS D'E. COLI Nous avons cherché à étudier les effets de NusG et NusA sur l'élongation de la transcription, individuellement ou simultanément. J'ai réalisé des essais de transcription sur deux types de pause différentes. Tout d'abord, j'ai reconstitué l'ARNP-EC au niveau de la pause his de E. coli, une pause bien caractérisée impliquée dans l'atténuation de l'opéron de biosynthèse de l'histidine. Lors de cette pause, l'ARNP est stabilisée par une tigeboucle d'ARN. J'ai mesuré les taux d'échappement à la pause (voir Méthodes pour plus de détails). Sous des concentrations saturantes de NusA, qui diminue les taux d'échappement de pause de 3 à 4 fois par rapport à l'ARNP seule, ce qui est cohérent avec les données bibliographique. En présence de NusG, la durée de la pause est légèrement plus courte que pour l'ARNP seule. Lorsque les deux facteurs sont inclus dans la réaction, le temps de pause est plus court qu'avec NusA seul, mais toujours légèrement plus long comparé à l'ARNP sans aucun facteur de transcription (Figure 49, comparer les courbes en jaune et noir). Ce résultat est cohérent avec nos données structurelles et suggère que l'effet de NusA dans la stabilisation de l'état de pause, peut être partiellement compensé par NusG. Pour clarifier si l'effet de réduction de la pause de NusG repose sur sa capacité à concurrencer NusA pour la liaison à FTH de l'ARNP, j'ai utilisé NusG-NTD. NusG-NTD a eu le même effet que la protéine entière et a augmenté les taux d'échappement à la pause his-pause, ce qui suggère que le domaine N-terminal de NusG seul est suffisant pour réduire l'effet de NusA. Il pourrait le faire en affectant le pivotement de l'ARNP par NusA et une tige-boucle d'ARN (Figure 49, comparer les courbes en violet, vert et jaune). v. EFFETS DE NUSA ET NUSG SUR L'O PS-PAUSE D'E. COLI En utilisant des essais de transcription (voir Méthodes pour plus de détails), j'ai testé les effets de NusA et NusG sur la pause ops. Il s'agit d'une pause régulatrice stabilisée par le retour en arrière de l'ARNP. L'ajout de FT n'a pas provoqué de changements majeurs dans la localisation du site de pause (Figure 50). L'ajout de NusG a diminué la pause, confirmant des résultats antérieurs (Figure 50, comparer les courbes en noir et rouge). NusA a augmenté de manière significative la fraction d'ARNP en pause et a prolongé sa durée (Figure 50, comparer les courbes en jaune et noir). Lorsque les deux facteurs sont présents à des concentrations saturantes dans la réaction, ils annulent leurs effets respectifs et la pause revient aux niveaux observés en l'absence de tout facteur de transcription (Figure 50, comparer les courbes en vert et noir). vi. EFFETS DE NUSA ET NUSG SUR LA TERMINA ISON DE RHO Nos résultats structurels suggèrent que NusG-CTD est libre de se lier à d'autres cibles lorsque NusA se lie à l'ARNP et occupe le module FTH. Il a été proposé que NusG-CTD stimule le facteur de terminaison Rho. NusA peut donc indirectement favoriser la terminaison par Rho par la libération de NusG-CTD du module FTH et ceci en augmentant la probabilité de pause. Pour vérifier si les deux facteurs peuvent coopérer pendant la terminaison dépendante de Rho, j'ai effectué des essais de terminaison l tR1 de la transcription en présence du facteur Rho purifié (Figure 51). Ce terminateur a été caractérisé pour avoir plusieurs sites de terminaison (précoce et tardif). Environ 13 % des ARNP peuvent terminer leurs transcriptions en l'absence de Rho, mais en sa présence environ 45 % d'ARNP vont terminer leur transcription. L'ajout de NusA réduit l'efficacité de la terminaison à 23 %, conformément aux rapports précédents. Tandis que NusG augmente légèrement la terminaison à 51 %. En présence des deux facteurs (NusA et NusG), l'efficacité de la terminaison atteint 56 % (Figure 52). Pour mieux comprendre, j'ai suivi la terminaison l tR1 précoce de la transcription au cours du temps. Afin d'être plus objectifs, nous avons modélisé la terminaison en utilisant un modèle cinétique simple. L'incapacité à saisir les points de mesure expérimentaux à un intervalle de temps très court fait de ce modèle un modèle mathématique limité, mais il montre une comparaison quantitative relative entre les différentes réactions. Ce modèle illustre qu'il y a deux possibilités pour une ARNP rencontrant un terminateur : elle peut soit interrompre la transcription et les produits d'ARN s'accumulent ; sois contourné le terminateur et reprendre la transcription. En pratique, la réaction se déroule comme un intermédiaire entre ces deux extrêmes. Ainsi, la fraction d'ARNP dans cette région de terminaison est l'addition des espèces terminées et des espèces restantes qui dépassent le terminateur. Une fraction de l'ARNP s'arrête brièvement dans la région de terminaison précoce, si Rho est présent, il a un effet modeste pour augmenter l'efficacité de la terminaison (Figure 54. A et E, comparer la courbe noire à la courbe bleue). L'ajout de NusA augmente fortement la fraction d'ARNP en pause, mais l'efficacité de la terminaison est comparable à celle de Rho seul. Ceci indique que la majorité d'ARNP en pause reprend la transcription (Figure 54. E, comparer la courbe bleue à la courbe jaune). L'ajout de NusG augmente l'efficacité de la terminaison et il semble que chaque ARNP en pause soit maintenant engagée pour la terminaison (Figure 54. E, comparer les courbes en noir, rouge et vert). De manière surprenante, un effet coopératif se produit en présence de NusA et NusG. Tout d'abord, la quantité d'ARNP en pause atteint un niveau similaire à la réaction en présence de NusA seul (probablement l'effet NusA), et ensuite presque toutes les ARNP terminent leur transcription (probablement l'effet NusG). Ceci permet d'augmenter l'efficacité de la terminaison globale de 6 fois par rapport à l'ARNP avec Rho (Figure 53). Il est important de noter que la délétion de NusG-CTD abolit cet effet (Figure 54. G). Dans la région tardive, un résultat légèrement différent émerge. L'ajout de Rho augmente l'efficacité de la terminaison de presque 4 fois. L'ajout de NusA augmente aussi la pause dans la région tardive, mais la terminaison apparaît beaucoup plus tard, en effet l'ARNP dépasse la région de terminaison tardive. NusG n'a pas d'effet important en soi, mais ensemble avec NusA, il engage tous les complexes en pause à se terminer. En conclusion, NusA et NusG peuvent coopérer pour augmenter l'efficacité au niveau précoce de la terminaison Rho-dépendante. NusG convertit toutes les fractions des ARNP en pause vers la terminaison. NusA peut avoir un double effet : (i) il augmente la fraction d'ARNP en pause qui est susceptible de se terminer lorsque NusG et Rho sont présent ; et (ii) il peut également augmenter la fraction de NusG-CTD disponible pour stimuler Rho en occupant le module FTH de l'ARNP. IV. DISCUSSION Les structures de l'ARNP au cours des différentes phases du cycle de transcription ont par le passé été associées à des conformations distinctes. La conformation pivotée récemment découverte a été proposée comme une complexe d'élongation en pause/inactif. J'ai observé qu'il s'agit d'un mouvement intrinsèque et naturel de l'ARNP. Dans cette étude, J'ai résumé certaines caractéristiques de la conformation pivotée : cette conformation empêche le rNTP d'entrer dans le site actif par le domaine SI3 connecté à un module de l'ARNP appelé en anglais triger loop ; elle réduit l'angle entre l'ADN en amont et l'ADN en aval de la bulle de transcription ; elle déstabilise l'hybride ARN-ADN en relâchant les contacts entre l'ARNP et l'hybride (cela peut ralentir la vitesse d'extension de l'ARN) ; elle élargit le canal de sortie de l'ARN (et par conséquent permet la formation de structures secondaires d'ARN à l'intérieur du canal de sortie) ; et elle fléchit une longue hélice alpha de l'ARNP, 'Brige helix' en anglais (BH). Quand BH est fortement plié, ceci pourrait empêcher le rNTP d'entrer dans le site actif et ainsi empêcher l'addition de nucléotide. Une description plus pertinente serait d'associer l'équilibre de ce mouvement avec la régulation de la pause, dans le cas où l'ARNP est en pause, l'équilibre est déplacé vers la conformation pivoté ; alors que dans le cas où elle est active, l'équilibre est déplacé vers la conformation non-pivotée. Ici, mes résultats ont montré que les FT NusG ou NusA règlent la transcription en ajustant cet équilibre. NusA favorise les pauses transcriptionnelles qui dépendent de l'adoption par l'ARNP de l'état pivoté, alors que NusG défavorise ces pauses. Bien que ces deux facteurs interagissent avec le module FTH de l'ARNP, ils peuvent se lier à elle simultanément. Ces deux observations (l'équilibre du mouvement et interaction avec le module FTH) expliquent leurs effets de compétition lors des pauses transcriptionnelles, cependant cela constitue également un avantage pour coopérer lors de la terminaison de la transcription. Lors de la terminaison de la transcription, NusA augmente la fréquence et la durée de la pause au terminateur, mais il délocalise la terminaison qu'il engendre sur les régions situées en aval. D'autre part, le FT NusG diminue l'efficacité de la pause, mais améliore la terminaison dans la région du terminateur précoce. En présence à la fois de NusA et de NusG, un effet cumulatif et coopératif sur l'augmentation de l'efficacité de la terminaison précoce a lieu. Nous proposons un modèle ou après l'initiation de la transcription et la sortie du promoteur, NusA et NusG s'approchent et se lient à l'ARNP. Ils influencent l'équilibre entre l'état pivoté et l'état non pivoté pour contrôler la mise en pause ou la sortie de la pause de l'ARNP. Une fois que le ribosome a rattrapé l'ARNP, NusG-CTD pourrait se lier au ribosome pour coupler la traduction à la transcription. Cela pourrait avoir pour effet de stabiliser NusG sur la EC de l'ARNP et donc d'empêcher une nouvelle pause et d'augmenter la vitesse de transcription. Lorsque l'ARNP est loin devant le ribosome, NusA peut augmenter la pause pour ralentir l'ARNP. Lorsque l'ARNP atteint les sites de terminaison, NusA occupe le module FTH de l'ARNP et augmente la fraction d'ARNP en pause. Le CTD libre de NusG active Rho et conduit les autres ARNP en pause vers la terminaison. V. CONCLUSION Lors de mes quatre années de thèse, dans l'équipe d'Albert WEIXLBAUMER, j'ai développé des compétences en biologie moléculaire, biochimie, bio-informatiques et traitement de données. Jusqu'à présent, mes travaux de doctorat ont conduit à résoudre trois structures de complexes d'élongation, correspondant à l'ARNP-NusA, l'ARNP-NusG et l'ARNP-NusA-NusG. Ainsi, grâce à cette étude, mes résultats montrent que les deux facteurs NusA et NusG se lient à plusieurs sites distincts sur l'ARNP. Cependant, j'ai observé un phénomène de compétition entre les sous-domaines des deux facteurs, de plus, ils peuvent influencer l'équilibre du pivotement et piloter l'ARNP vers l'état de pause ou l'état actif au cours de l'élongation. Cette compétition est cohérente avec les résultats biochimiques que j'ai obtenus. En outre, ces observations structurales ont suscité de nouveaux questionnements : quel est l'effet de la combinaison de NusA et NusG sur les pauses et la terminaison de la transcription ? J'ai émis l'hypothèse que pendant l'élongation, ces facteurs peuvent avoir des effets antagonistes sur les pauses. Cependant, ils peuvent aussi coopérer avec le facteur de terminaison Rho pour terminer la transcription selon les tests de pause et de terminaison que j'ai réalisé.

Figure 1 :

 1 Figure 1: Crystal structure of the Thermus thermophilus RNAP. RNA polymerase (core enzyme) elongation complex. The different subunits are labelled and coloured separately. (pdb: 2O5J, resolution: 3 Å.)

Figure 2

 2 Figure 2 Bacterial promoter DNA and its interaction with RNAP and σ factor. A) The cryo-EM structure of the holoenzyme in E. coli. The different subunits are labelled and coloured separately. (pdb: 6C9Y) B) Schematic diagram showing the structure of the promoter in bacteria including the UP element, the -35 element, the extended -10 element (Ext), the -10 element and the discriminator element (Dis). The CTD of the α subunit of RNAP (αCTD) and four domains (domains 4-1) of the σ factor interact with these promoter elements; adapted from (Browning and Busby, 2016; Haugen et al., 2008). C) A crystal structure of open complex in T. aquaticus containing holoenzyme and promoter DNA which includes the full transcription bubble. The different subunits are labelled and coloured separately in the figure. (pdb: 4XLN)

Figure 3 :

 3 Figure 3 : The 'DNA-scrunching' model proposed for transcription initiation.

Figure adapted from

  Figure adapted from[START_REF] Kapanidis | Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism[END_REF]

Figure 4 H

 4 ) is a smaller, funnel-shaped channel leading from the RNAP surface to the active site and separated by BH and TL from the primary channel. It is thought to allow the exchange of rNTP substrates and pyrophosphate. The downstream DNA is held by the β-lobe and the shelf (Figure 4 A). The clamp domain (Figure 4 A), sits on the shelf domain. At the upstream end of the RNA-DNA hybrid, the RNA chain extends to the surface of RNAP through the RNA exit channel (Figure 4 G), which is clasped by the clamp and β flap domain. The β flap is on the opposite side of the clamp, a small helix on the top of the β flap, called the flap-tip-helix (FTH) is extremely flexible and often fluctuates across the top of the RNA exit channel (Figure 4 G). It has been hypothesized that the FTH may interfere with nascent RNA folding, and pulling the FTH away from the top of the RNA exit channel might favour RNA folding and transcription

Figure 4 :

 4 Figure 4 : Structure of elongation complex with its active site.

Figure 5 :

 5 Figure 5 : Nucleotide addition during elongation. A scheme of nucleotide (NTP) addition cycle (Left) and the nucleotide addition reaction in the active site (right). POST: posttranslocated state; PRE: pre-translocated state. The Mg2+ I and Mg2+ II are labelled by '1' and '2' in yellow circles (right). The figure on the right is adapted to Belogurov and Artsimovitch (Belogurov and Artsimovitch, 2019).

  It has two conformations, the open-ring and the closed-ring states (Figure 6) (Ray-Soni et al., 2016).

Figure 6 :

 6 Figure 6 : Structure of hexamer ρ factor. Hexamer ρ shows open-ring state (left) and closed-ring state (right). The figure is adapted from Ray-Soni et al. (Ray-Soni et al., 2016)

  HO Some debates exist for how ρ triggers termination. The classical model suggests that ρ can interact with unstructured rut RNA through the primary RNA binding sites on the ρ-NTD under the 'open-ring' conformation state. RNA is then loaded and gets bound to the secondary RNA binding sites inside the ring on the ρ-CTD. Thus, it was proposed when the ring is closed, ρ could translocate along RNA to catch up with the paused RNAP and remove an RNAP EC to terminate the transcription (Ray

Figure 7 :

 7 Figure 7 : The comparison of two termination models. Comparison of the classical models: transcription termination by termination factor ρ (Ray-Soni et al., 2016) and recent model based on high resolution structures (Said et al., 2020). Figures were adapted fromthe articles.

  EN T P A U S I N G P HE N O M E N O N D U R I N G E L O N G A T I O N Pausing probably plays the most important role during the elongation phase of transcription. Paused RNAP has been detected as halted complexes at distinct sites for variable durations. Pause escape occurs when the active site rearranges itself into an active configuration and resumes nucleotide addition. Recent studies identified around 20000

  S S I I P A U S E : B A C K T R A C K I N G -S T A B I L I Z E D P A U S I N GThe class II pause depends on and is stabilized by RNAP backtracking (reverse translocation relative to the DNA template) and can be triggered by weak AT(U)-rich DNA-RNA hybrids or an incorrect rNTP introduced during transcription (which also weakens the thermodynamic stability of the hybrid). This instability drives RNAP to back-translocate along the DNA to form a more stable RNA-DNA hybrid.The 3'-end of the RNA from the A site is extruded into the secondary channel of the RNAP. The active site and the secondary channel are blocked by the RNA, which renders RNAP inactive and inaccessible to rNTPs. To return to the active state, RNAP needs to free the active site and the secondary channel either by forward translocation along the DNA or by cleavage of the RNA so a new, shorter RNA aligns its 3'-end

Figure 8

 8 Figure 8 The consensus sequence of transcriptional pausing. Sequencing readout of the pausing sequence (top) around the transcriptional bubble (bottom). Figure adapted from Vvedenskaya et al. (Vvedenskaya et al., 2014).

  divide it into 3 parts: the N-terminal domain (NusA-NTD), the C-terminal domain (NusA-CTD) further sub-divided into two acidic repeat domains (AR1, and AR2), and an intermediate domain. The intermediate domain is subdivided into RNA binding domains called S1 (named after ribosomal protein uS1), KH1, and KH2 (K-homology domain named after

  S A B I N D I N G T O RNAP NusA binds RNAP near the RNA exit channel with 4 different contact points. The most important contact between RNAP and NusA is established by the NusA-NTD with the α2-CTD of RNAP and the flap-tip-helix (FTH) in the β-flap domain. The β-flap domain of RNAP is positively

  at least two domains: an NTD and a CTD, with a flexible linker connecting them. The NusG N-terminal domain (NusG-NTD) contains the NGN motif (NusG N-terminal), which is universally conserved and also found in eukaryotic Spt5 proteins. The NusG-NTD binds to RNAP through mostly hydrophobic interactions(Kang et al., 2018a). It bridges the active site cleft by interacting with the clamp helices (CH) of the β' subunit of RNAP and the protrusion and gate loop (GL) of the β subunit of RNAP. It has been suggested that NusG acts as a lock between the β and β' subunit to prevent backtracking and keep RNAP in the active conformation(Kang et al., 2018a;[START_REF] Sevostyanova | The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG[END_REF].The CTD of NusG folds into a five-stranded anti-parallel β sheet and looks like a clip. It embeds a Kyrpides, Ouzounis, Woese (KOW) motif. This motif is suggested to interact with either protein or nucleic acid.Two well-known interaction partners are ρ and the ribosome. The short flexible linker connecting the CTD and NTD of NusG allows NusG to act as a bridging molecule, helping to connect RNAP to diverse types of machineries and accomplish multiple tasks such as RNA processing, translation and antitermination modification[START_REF] Nandymazumdar | Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins -Shifting shapes and paradigms[END_REF].

  2.2 M E C HA N I S M O F S U P P R E S S I N G P A U S I N G R EG U L A T I O N W I T H N U S G

  2018, Kang et al. published two reconstructions of RNAP paused at the ops-site bound to either NusG or RfaH (NusG-opsEC or RfaH-opsEC) (Kang et al., 2018a). RfaH is a paralogue of NusG and belongs to the NusG SP family. Both are elongation factors that prevent pausing during transcription but RfaH is restricted to a subset of bacterial operons. The comparison of these two structures helped us to understand their roles. According to Kang et al., NusG and RfaH suppress pausing using three different principles. First, the NusG-NTD might stabilize the first re-annealed bp of the upstream DNA duplex at position -10 bp immediately upstream from the fork junction and the RNA-DNA hybrid, as shown by cross-linking experiments and Exonuclease III footprinting(Kang et al., 2018a;

A

  simple model, which nevertheless recapitulates the basic mechanism, is the bacterial RNAP. It allows us to understand the essential functions and provides basic mechanistic insights into the very complex process of transcription. The two essential bacterial transcription factors NusG and NusA are both highly conserved. Using ChIP-chip, Landick and co-workers suggested that after the initiation of transcription these two TFs might bind simultaneously to an elongating RNAP[START_REF] Mooney | Regulator Trafficking on Bacterial Transcription Units In Vivo[END_REF]. Despite the numerous previous studies, the mechanism of RNAP regulation by NusG and NusA is still not fully understood. For example, how does NusG increase transcription rates and prevent pausing? How does NusA regulate RNAP when the enzyme is not paused? How do these two transcription factors regulate RNAP at the same time during elongation as well as transcription termination, and how is this compatible with their opposing effects? I performed structural studies using single particle cryo-EM on three elongation complexes: RNAP bound to NusG only, RNAP bound to NusA only, and RNAP bound to both factors simultaneously. The combination of structural information with detailed biochemical studies will further improve our understanding on the role of these two proteins.Moreover, there is an ongoing debate about the role of NusG and whether or not it serves as a bridge between transcription and translation. Additionally, my expertise on NusG allowed me also to contribute to the expressome project, which had several objectives including to study if and how NusG links RNAP and the ribosome.

  MODEL E. coli TOP10 cells (Invitrogen) were used for plasmid construction. E. coli LACR II (Low Abundance of Cellular RNases), a derivative of E. coli LOBSTR (Andersen et al., 2013), was constructed and used for recombinant protein expression. LACR II is a double knock-out for two of the most abundant RNases (RNase I and RNase II, genes rna rnb) to decrease the amount of RNase contamination in the purified protein sample.

  i. PURIFICATION OF NUSG/NUSG-NTD pSKB2_His6_HRV3C_Eco_nusG_FL or pAX0_His10_HRV3C_Eco_nusG_NTD plasmids were constructed to encode E. coli NusG-FL or NusG-NTD containing His6-/His10-tags at the N-terminus followed by an HRV3C cleavage site (PreScission protease), respectively. NusG encoding plasmids were transformed into E. coli LACRII (rna-rnb-) strain. NusG-FL and NusG-NTD were separately overexpressed in 6 L LB culture (50 μg/ml kanamycin), induced by 1 mM IPTG at an OD600nm of 0.6 for 3 hours at 37°C. The cells were harvested by centrifugation (30 min 4000 rpm at 4°C (JLA 8.1000 Beckman rotor)), resuspended in 4 volumes of lysis buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 233 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine, cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 1 tablet/50 mL)), and lysed by sonication (Sartorius, BBI-8535108, LABSONICRP, amplitude 60%, 2 min with pulse-on/off durations of 0.5 sec, repeated 5 times with cooling in icewater bath in between). The lysates were cleared by centrifugation at 40,000 g for 30 minutes at 4°C in a 50.2 Ti rotor (Beckman Coulter). The nucleic acids and their interacting proteins were precipitated by adding 0.6% of polyethyleneimine (PEI) and removed by centrifugation at 45,000 g for 20 minutes at 4°C. Ammonium sulfate was added to the supernatant to a final concentration of 0.37 g/mL and the precipitate was collected by centrifugation at 45,000 g for 20 minutes at 4°C. The pellet was then resuspended in IMAC (Immobilized Metal ion Affinity Chromatography) buffer A (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM imidazole, 1 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine). NusG proteins were first purified by passing the supernatant over a 5 mL HiTrap IMAC HP column (GE Healthcare) charged with NiSO4 and eluted at 200 mM imidazole using a step gradient into buffer B (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole, 1 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine). The peak fraction was then dialyzed overnight against dialysis buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5% glycerol, 1 mM β-mercaptoethanol) in the presence of HRV3C PreScission protease (1 mg HRV3C per 18 mg of protein). The dialyzed sample was reloaded on the IMAC column to separate it from uncleaved protein and His-tagged HRV3C. Cleaved NusG binds weakly to the IMAC column and was eluted at around 60 mM imidazole, and further dialyzed into ion-exchange buffer A (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA pH 8.0, 5% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine). The protein was then loaded on a 5 mL HiTrap Q HP column (GE Healthcare) and eluted using a gradient of 0-100% ion-exchange buffer B (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.1 mM EDTA pH 8.0, 5% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine) over 20 column volumes (CVs) to elute NusG. The peak was concentrated and further purified by gel filtration using a Superdex 75 16/600 (GE Healthcare) column equilibrated with GF buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine). The final protein was concentrated to 5 mg/mL, aliquots were flash frozen, and stored at -NusA with a His10-tag at the N-terminus was overexpressed in E. coli BL21 (rna-rnb-) strain (a generous gift from the Deutscher lab, University of Miami, Miami, USA). From a 6 L LB culture (50 mg/L kanamycin), expression was induced with 1 mM IPTG at an OD600nm of 0.7 for 3 hours at 37°C. Bacteria were collected by centrifugation (30 min 4000 rpm at 4°C (JLA 8.1000 Beckman rotor)), resuspended in 5 volumes of lysis buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine, DNase I (0.5 mg/250 g cell), cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 1 tablet/50 mL)) and lysed using sonication (amplitude 60%, 2 min at pulse on/off durations of 0.5 sec, repeated 5 times with cooling in ice-water bath in between (Sartorius, BBI-8535108, LABSONICRP)). The lysates were cleared by centrifugation at 40,000 g for 30 minutes and the supernatant was loaded on two 5 mL HiTrap IMAC HP columns (GE Healthcare) and eluted using a linear gradient into lysis buffer containing 250 mM imidazole over 10 CVs. Histagged HRV3C PreScission protease was added to peak fractions containing NusA and dialyzed overnight against lysis buffer with 50 mM NaCl. Cleaved NusA was separated from non-cleaved protein, and the His-tagged protease by reloading on the IMAC column. The flowthrough was applied to a 5 mL HiTrap Q HP column (GE Healthcare).

  pACYC_Duet1_rpoZ was co-transformed to avoid sub-stoichiometric amounts of the RNAP ω subunit. E. coli RNAP was overexpressed in 6 L LB culture (50 μg/mL kanamycin, 34 μg/mL chloramphenicol), which was induced by adding 1 mM IPTG at an OD600nm of 0.6-0.8 and incubated at 37°C for 2 hours. After centrifugation (30 min 4000 rpm at 4°C (JLA 8.1000 Beckman rotor)), the cell pellet was resuspended in 5 volumes of lysis buffer (50 mM Tris-HCl pH 8.0, 5% glycerol, 1 mM EDTA pH 8.0, 10 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 μM ZnCl2, 100 mg DNase I, cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 1 tablet/50 mL)) followed by sonication (amplitude 60%, 2 min at pulse on/off durations of 0.5 sec, repeated 5 times with cooling in ice-water bath in between (Sartorius, BBI-8535108, LABSONICRP)). The insoluble material and cell debris was removed by centrifugation (40,000 g, 30 minutes, 4°C), and RNAP was isolated from the supernatant by PEI precipitation (0.6% final concentration). The PEI precipitate was washed twice with 100 mL PEI wash buffer (10 mM Tris-HCl pH 8.0, 5% glycerol, 0.1 mM EDTA pH 8.0, 0.5 M NaCl, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 μM ZnCl2) and RNAP was extracted three times with 10-20 mL PEI elution buffer (10 mM Tris-HCl pH 8.0, 5% glycerol, 0.1 mM EDTA pH 8.0, 1 M NaCl, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 μM ZnCl2). RNAP was precipitated using ammonium sulfate (~35mg/100ml). The precipitate was collected by centrifugation (10,000 g, 10 minutes, 4°C) and resuspended in IMAC buffer A (20 mM Tris-HCl pH 8.0, 1 M NaCl, 5% glycerol, 5 mM βmercaptoethanol, 0.1 mM PMSF, 1 mM benzamidine, 10 mM ZnCl2) and passed over a 20 mL Ni-IMAC Sepharose HP column (GE Healthcare) using a step gradient with 250 mM imidazole in IMAC buffer B for the elution (2 CVs IMAC buffer A, 5 mM imidazole wash for 2 CVs, gradient from 5 to 40 mM imidazole over 1 CV, 40 mM imidazole for 5 CVs, and final elution step at 250 mM Imidazole). In order to cleave the affinity tag, peak fractions were pooled and dialyzed in the presence of HRV3C PreScission protease (1 mg HRV3C per 8 mg of protein) overnight in dialysis buffer (20 mM Tris-HCl pH 8.0, 1 M NaCl, 5% glycerol, 5 mM βmercaptoethanol, 10 μM ZnCl2). The uncleaved/cleaved RNAP, the His10-tag, and HRV3C were separated by reloading the sample on the IMAC column. The cleaved RNAP was then dialyzed into Bio-Rex buffer (10 mM Tris-HCl pH 8.0, 5% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 μM ZnCl2) until the conductivity was £ 10 mS/cm. The RNAP was then loaded on a 50 mL Bio-Rex 70 column (BIO-RAD) and eluted using a linear gradient into Bio-Rex buffer plus 1 M NaCl over 5 CVs. Afterwards, the concentrated peak was purified by gel filtration using a HiLoad Superdex 200 PG 26/600 column (GE Healthcare) equilibrated with GF buffer (10 mM HEPES pH 8.0, 0.5 M KCl, 1% glycerol, 2 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 10 μM ZnCl2, 1 mM MgCl2). In the end, the protein was dialyzed into EM buffer (10 mM HEPES pH 8.0, 150 mM KOAc, 2 mM DTT, 10 μM ZnCl2, 5 mM Mg(OAc)2), concentrated to ~80 mg/mL, aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.iv. PURIFICATION OF E. COLI TRANSCRIPTION TE RMINATION FACTOR RHOThe pET28B plasmid containing the gene for termination factor ρ was transformed and overexpressed in E. coli BL21-DE3 (pLysS) strain. In 6 L of LB culture (50 μg/mL kanamycin and 37 μg/mL chloramphenicol), expression was induced with 0.8 mM IPTG at OD600nm of 0.6-0.8 for 3 hours at 37°C. Bacteria were harvested by centrifugation (30 min 4000 rpm at 4°C (JLA 8.1000 Beckman rotor)), resuspended in 5 volumes of lysis buffer (50 mM Tris-HCl pH 7.5, 5% glycerol, 5 mM EDTA pH 8, 0.1 mM DTT, 100 mM NaCl, 10 mM imidazole, 1 mM β-mercaptoethanol, 1 mM PMSF, 1 mM benzamidine, cOmplete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 1 tablet/50 mL) and 0.01 volume of 5% sodium deoxycholate) and lysed using sonication (amplitude 60%, 2 min at pulse on/off durations of 0.5 sec, repeated 5 times with cooling in icewater bath in between (Sartorius, BBI-8535108, LABSONICRP)). DNase I (0.5 mg/250 g cells) and 24 mM MgCl2 were added to the sample and incubated for 20 minutes on ice. The lysates were cleared by centrifugation at 30,000 g for 30 minutes at 4°C (50.2 Ti rotor, Beckman Coulter). 5% v/v polyethyleneimine was added to the supernatant and the precipitate was removed by centrifugation at 30,000 g for 15 minutes at 4°C. 0.5 g/mL of ammonium sulfate was added to the supernatant. After one hour on ice, the precipitate was collected by centrifugation at 30,000 g for 20 minutes at 4°C. The pellet was resuspended in and dialyzed overnight against buffer A (10 mM Tris-HCl pH 7.6, 5% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 100 mM NaCl). ρ was first purified by passing the supernatant through a 20 mL SP sepharose column (GE Healthcare) using a 0-60% linear gradient of buffer B (10 mM Tris-HCl pH 7.6, 5% glycerol, 0.1 mM EDTA pH 8, 0.1 mM EDTA pH 8, 0.1 mM DTT, 1 M NaCl) over 11 CVs. After adjusting the salt concentration of the pooled-peak fraction to less than 400 mM (by dilution if necessary), it was loaded onto a 5 mL Heparin HP column. The column was equilibrated with Buffer A, and ρ was eluted using a 0-100% linear gradient into buffer B over 20 CVs. The peak was concentrated and further purified by gel filtration using a Superdex 200 16/60 column (GE Healthcare) equilibrated with GF buffer (20 mM Tris-HCl pH 7.9, 5% glycerol, 0.2 mM EDTA pH 8, 500 mM KCl, 0.2 mM DTT). The peak fractions containing ρ were then dialyzed against storage buffer(10 mM Tris-HCl pH 8, 50% glycerol, 0.1 mM EDTA pH 8, 150 mM KCl, 1 mM DTT), concentrated to 80 mg/mL, flash frozen, and stored at -80°C.VI. TESTS BEFORE PROTEIN PURIFICATIONi. EXPRESSION TEST An overnight culture of E. coli LACR II cells carrying pSKB2_Eco_nusG was used to inoculate 20 mL of LB media containing 50 μg/ml kanamycin. The cultures were grown at 37°C with agitation at 200 rpm (Excella E24). The starting OD600nm of the culture was 0.1 by using the appropriate amount of overnight culture. When the culture reached an OD600nm of 0.6-0.7 protein expression was induced with 0.5 mM IPTG, and maintained for 3 hours at 37°C or overnight at 18°C. To confirm protein expression, 20 μL of samples were taken before and after induction and normalized based on the OD600nm reading. These samples were centrifuged at 7000 g for 2 min, after removal of the supernatant the cells were resuspended in 20 μL of 5× SDS loading dye (250 mM Tris HCl 4% SDS, 0.1% bromophenol blue, 20% glycerol, 250 mM DTT) and were boiled at 98°C for ~5 to 10 min before loading on 10%, 15% or 20% SDS polyacrylamide gel. Proteins were visualized by Coomassie brilliant blue staining (Coomassie Brilliant Blue R-250

  iii. BINDING TESTS OF TAGGED PROTEINS TO MA TRIX This binding test protocol is a common procedure for all proteins with His-tags. Buffer A used to lyse the cells and test binding to affinity matrices can be adjusted for each specific protein. After solubility test, the IMAC matrix (Perfect Pro Ni-NTA Superflow, Biotech) was washed once with water and twice with buffer A by centrifugation at 400 g for 2 min, (50 μL of dry Ni-NTA beads corresponds to about 100 μL of Ni-NTA solution and washed with 1ml of buffer). The buffer equilibrated matrix was distributed into tubes and was mixed with 1 mL of the supernatant for the solubility test, and left at 4°C with gentle agitation for 1-2 hours. After centrifuging at 25,000 g for 2 min, the sedimented Ni-NTA beads were washed with 500 μL buffer A to remove unbound protein. Finally, the flow through and the IMAC beads were mixed with 5× SDS loading dye and boiled at 98°C for 5~10 min before loading on an SDS polyacrylamide gel.VII. LABELLING OF RNA OR DNA OLIGONUCLEOTIDESIn a 1.5 mL tube, the components were prepared to a final concentration of 5 μM oligonucleotide (chemically synthesized RNA or DNA), 5 μL 32P-γ-rATP (1.66 pmol/μL), 10 U T4 PNK (polynucleotide kinase), 1× PNK buffer (Thermo Scientific) in a 20 μL final volume. After incubation for 1 hour at 37°C, cold 2.5 μM rATP (non-radioactive) was added to drive the reaction to completion, PNK was inactivated by incubation at 65-70°C for 20 min. Free 32P-γ-rATP was removed using a desalting column (Biorad spin columns filled with ~200 μL of Sephadex G50-matrix; typically, the matrix volume was chosen to be 10 times larger than the sample volume). The resin was loaded into the column and washed twice with 200 μL water by centrifugation at 2000 g for 1 min. 20 μL of labelling reaction was then carefully loaded on the resin, and the labelled oligo was collected from the flow-through by centrifugation at 2000 g for 1 min. Labelled nucleic acids were kept at -20°C until use. VIII. RECONSTITUTION OF NUCLEIC ACID SCAFFOLD (RNA:T-DNA:NT-DNA) Unlabelled nucleic acid scaffolds (short DNA and RNA oligonucleotides annealed to mimic a transcription bubble with nascent RNA) were prepared in a volume of 100 μL by mixing 50 μM RNA, 100 μM template DNA (t-DNA) and 100 μM non-template (nt-DNA) in reconstitution buffer (RB; 10 mM Tris HCl pH 8.0, 40 mM KCl, 5 mM MgCl2). Scaffolds were assembled using a thermocycler (program: 95°C 2 min, 75°C 2 min, 45°C 5 min, 45°C to 4°C at a rate of -2°C/2 min, hold at 4°C) and stored at -20°C.Three different types of radioactively labelled scaffolds (32P) were used: only labelled RNA; only labelled t-DNA; only labelled nt-DNA. Samples containing only RNA and t-DNA were first incubated at 98°C for 2 min for denaturation, and then the tube was kept in a water bath starting at 85°C, which slowly cooled down to 25°C. The nt-DNA was added only after the RNA:t-DNA complex was first incubated with RNAP. We found this improved the fraction of labelled RNA incorporated into active RNAP ECs. The concentration of components for each scaffold was 2.5 μM 32P-RNA and 5 μM t-DNA and nt-DNA; or 1.25 μM RNA, 2.5 μM 32Pnt-DNA and 2.5 μM t-DNA; or 1.25 μM RNA, 2.5 μM 32P-t-DNA with 2.5 μM nt-DNA. Nucleic acid scaffolds were stored at -20°C.

  using a final concentration of 0.5 μM of 32P-labeled RNA, 1 μM of template DNA (tDNA+ntDNA) in elongation buffer (20 mM Tris HCl pH 8.0, 20 mM NaCl, 15 mM MgCl2, 150 μM EDTA pH 8.0, 1 mM DTT, 0.02 mg/mL BSA and 1 μM RNAP). Each reaction contained only one nucleotide: rATP, rCTP, rGTP or rUTP with a final concentration of 10 μM to monitor if specific incorporation of only one nucleotide occurs.The reaction mixtures were incubated at 37°C for 5 minutes. The samples were loaded on a 20% denaturing polyacrylamide gel (20% polyacrylamide and 7 M urea; 1× TBE), and the gel was run in TBE buffer (890 mM Tris HCl pH 8.0, 890 mM boric acid, 20 mM EDTA pH 8.0) until the upper dye was near the bottom of the gel at 50 W. An X-ray film was used to support the gel and it was exposed using a storage phosphor screen overnight at -80°C and visualized using a PhosphorImager (Typhoon, GE Healthcare).

I

  constructed an initial model of the different complexes by combining the cryo-EM structure of E. coli RNAP (PDB ID: 6ALH)(Kang et al., 2018), a previously published structure of a paused E. coli RNAP (PDB ID: 6FLQ)[START_REF] Guo | Structural Basis for NusA Stabilized Transcriptional Pausing[END_REF] and the NMR structure of E. coli NusG (PDB ID: 2K06)[START_REF] Mooney | Two Structurally Independent Domains of E. coli NusG Create Regulatory Plasticity via Distinct Interactions with RNA Polymerase and Regulators[END_REF]. UCSF Chimera(Pettersen Ef Fau -Goddard et al., 2004) was used for initial model placement into the EM maps. Model building included real space refinement using the Phenix software suite[START_REF] Discussion 9 | PHENIX : a comprehensive Python-based system for macromolecular structure solution[END_REF] and manual modifications in Coot(Emsley &

Figure 9

 9 Figure 9 Schematic of cloned nusG constructs.The nusG gene (thick red arrow) is cloned in frame with an Nterminal His10-tag (thick purple arrow) and HRV3C protease cleavage site (thick pink arrow). The construct contains a T7 promoter (thick white arrow) and T7 terminator (white rectangle) for expression in E. coli. RBS: ribosome binding site.

Figure 10 E

 10 Figure 10 E. coli NusG expression analysis. NusG-FL (left) or NTD (right) were overexpressed in E. coli LACR II cells. Cell lysate samples from different expression conditions (indicated on the top) were analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. The first lane represents respective cultures before induction, the second lane represents the culture induced at OD600nm of 0.6-0.8 with 0.5 mM IPTG at 37°C for 3 hours; the third lane represents the overnight (ON) expression at 18°C both induced at OD600nm of 0.6-0.8 with 0.5 mM IPTG. The band corresponding to NusG-FL (22 kDa) and NusG-NTD (15 kDa) are indicated with red stars. L: Ladder; Tmp: temperature.

Figure 11 Figure 12

 1112 Figure 11 Expression test of T. th or T. aq NusG-FL or NTD in E. coli LACR II. Different conditions (indicated on the top) were analyzed by 15% SDS-PAGE and stained with Coomassie brilliant blue. Left gel: lane 1, NusG-FL before induction; lane 2, NusG-NTD before induction; lane 3, T. th NusG-FL culture induced with 0.5 mM IPTG at OD600nm of 0.6-0.8 for 3 hours at 37°C; lane 4, T. th NusG-NTD culture induced with 0.5 mM IPTG at OD600nm of 0.6-0.8 for 3 hours at 37°C. Right gel: lanes 1-6, lysates before induction; lane 7, ladder; lanes 8-13, T. aq NusG cultures induced with 0.5 mM or 1mM IPTG at OD600nm of 0.6-0.8 for 3 hours at 37°C or overnight at 18°C. L: Ladder; FL: full length NusG; NTD: N-terminal domain of NusG; Tmp: temperature. The band corresponding to NusG-FL and NusG-NTD are indicated by red stars.

Figure 13

 13 Figure 13 Purification of NusG. L: Ladder. Lane 1: total cell lysate before induction; Lane 2, E. coli NusG-FL culture induced with 0.5 mM IPTG at OD600nm of 0.6-0.8 for 3 hours at 37°C; Lane 3, supernatant of cell lysate; Lane 4, pellet of cell lysate; Lane 5, sample purified by IMAC; Lane 6, sample after overnight dialysis and tag cleavage by HRV3C protease; Lane 7, sample purified on Q-column; lane 8, NusG purified by size-exclusion chromatography.

(

  NusG, NusA and RNAP) could form a complex in vitro. The Electrophoretic mobility shift assay (EMSA), is a simple and quick method that is often used to study macromolecular interactions. It can separate individual proteins or their complexes according to their size, charge and shape as they migrate through the gel at different rates. A transcription elongation complex contains RNAP, a nucleic acid scaffold (DNA and RNA), and possibly transcription factors (TFs). The nucleic acid scaffolds incorporated in the complex contain several mismatches between the non-template DNA (nt-DNA) and the template DNA (t-DNA) to mimic the transcription bubble. This artificial bubble also facilitates RNA annealing and RNAP binding (Figure15).

Figure 15

 15 Figure 15 Schematic of the DNA/RNA scaffold. Schematic of the DNA/RNA scaffold used for gel shifts. Different colours represent each component in the scaffold, non-template DNA (nt-DNA, lavender), template DNA (t-DNA, violet), and RNA (green). The DNA oligonucleotides contain mismatches in the central portion to mimic a transcription bubble and favour RNA annealing.

Figure 16

 16 Figure 16 Gel shift assay for detection of E. coli RNAP-scaffold-NusG complex. The gel was first stained with Coomassie brilliant blue (gel on the left), then with ethidium bromide (gel on the right). The different components in the samples are indicated on the top of the gel. The red stars indicate the NusG-EC.

Figure 18

 18 Figure 18 Verification of the scaffold binding to RNAP by radioactive labelling on native 2% agarose gel.

I

  monitored the transcription process with a series of time-point samples in presence or absence of NusG. The enlarged section of the gel (Figure 19 C) shows pause-supressing effect of E. coli NusG. This can be demonstrated by the 2-fold lower amount of paused species in presence of NusG (14% RNA species of total RNA at this time point for RNAP alone, and 7% for RNAP plus NusG). NusG also slightly increased pause-escape rates (shown by the rate of the fast species and the halflife of the reaction) (Figure 19 E).

Figure 19

 19 Figure 19 Transcription assay in presence of NusG.A) DNA template for transcription assays. Linear template DNA was generated by PCR amplification from a vector containing a T7A1 promoter (transcription start site indicated by bent arrow), a Uless region ending at A48, the E. coli ops-pause site, and a transcription terminator (release site at +217). B) A representative gel of a transcription assay (here RNAP alone is shown). Each lane of the 10% polyacrylamide urea gel corresponds to different time points during the transcription reaction and visualizes the transcripts generated from the template at 10'', 20'', 30'', 1', 1'30'', 2', 3', 5', and 10', as well as a chase reaction with high substrate concentration. The time point 0, corresponds to the halted A48 elongation complex (please note that some readthrough occured). Positions of the halted (A48) and paused (ops U60) transcripts are marked with red arrows. The vertical line indicates terminated transcripts. C) Enlarged section of the gel at the ops-site in absence or presence of NusG. D) Quantification of paused RNA products at U60. RNAP with NusG is shown in red, RNAP alone in black. E) The kinetic parameters were obtained from fitting the experimental data in D) using the equation shown above.

Figure 20

 20 Figure 20 One base pair extension assay on a scaffold. A) Schematic of the DNA/RNA scaffold used for the RNA extension assay with the 3'-terminal RNA nucleotide G14 and the incoming nucleotide C15 indicated. Different colours represent each component in the scaffold, non-template DNA (nt-DNA, lavender), template DNA (t-DNA, violet), and RNA (green). The DNA oligonucleotides contain mismatches in the central portion to mimic a transcription bubble and favour RNA annealing. B) RNA extension assay. The 15% polyacrylamide urea gel is revealed by phosphor-imager. RNA is labelled on the 5'-end by phosphorylation with 32P isotope. Then samples of the elongation reaction were taken at different time points indicated on the top of the gel. G14 is the 3'-terminal nucleotide at the beginning of the reaction and the incoming nucleotide is C15. C) Negative controls. Same RNA extension assay with different substrates is prepared. The substrate is indicated on the top of the gel: rATP, rCTP, rGTP or rUTP. Specific incorporation occurred only in the presence of rCTP. The first lane is the negative control and does not contain any nucleotide.

For

  structural studies I reconstituted RNAP EC on a nucleic acid scaffold that supports formation of a canonical post-translocated state. Then I added NusG (NusG-EC) or NusA (NusA-EC) or both transcription factors (NusA-NusG-EC) to form functional complexes (Figure 21 A). To obtain homogenous samples, I purified the complexes by size-exclusion chromatography. As shown on Figure 21, these complexes are stable during the purification.

Figure 21

 21 Figure 21 Reconstitution of homogeneous ECs on a canonical post-translocated scaffold. A) Schematics of the scaffold used for structural studies. Different colours represent each component in the scaffold, non-template DNA (nt-DNA, lavender), template DNA (t-DNA, violet), and RNA (green). The upstream and downstream DNA regions are highlighted in light blue. B) NusA-NusG-EC was assembled in vitro using an excess of NusA, NusG and scaffold. There are several peaks on the chromatogram. They correspond to the NusA-NusG-RNAP complex, the residual NusA, scaffold and NusG. The peak fraction containing the NusA-NusG-EC was analysed on 15% SDS-PAGE gel stained with Coomassie brilliant blue. The components of the complex are labelled on the right side of the gels. C) NusG-EC purified as in B). The NusA-EC was purified in the same way and behaved identical to the NusA-NusG-EC or NusG-EC (data not shown)

Figure 22

 22 Figure 22 Overview of consensus refinement of NusG-EC. A) A locally sharpened cryo-EM density map of the NusG-EC with nominal resolution 3.8 Å (on the left shown with a transparent map). The backbone is shown as ribbons coloured by chain. RNAP subunits are in different shades of blue, nt-DNA and t-DNA are in lavender and violet, RNA in green and NusG in red. B) A zoom-in at the active centre of RNAP, showing the DNA/RNA hybrid in the post-translocated state. C) Angular distribution plot shows random particle orientation of the NusG-EC reconstruction. D) Fourier shell correlation (FSC) plot for half-maps of the NusG-EC reconstruction. The final nominal resolution is estimated to be 3.8 Å using the gold-standard Fourier shell correlation criterion.

Figure 25

 25 Figure 25 Contact points between NusG and RNAP.

Figure 26

 26 Figure 26 The distance between the SI3 (β'945 -β'1130) domain and the β-lobe. The distances vary in NusG-EC (violet), NusG-opsEC (red) and λN-TAC (green): they are 17.5 Å, 18 Å and 18.7 Å, respectively.

Figure 27

 27 Figure 27 Heterogenous refinements of NusG-EC in CryoSPARC. The refined consensus structure from all particles (shown in grey on the top) has the highest resolution. The classes obtained from heterogeneous refinements are shown in colour. 'w/oG swi-' class in pink-orange has very weak density for NusG. 'w/oG swi++' class in purple lacks density for the upstream DNA and NusG. Three other classes correspond to NusG-EC but with different extents of swivelling of RNAP, 'w/G swi-' class in blue, 'w/G swi+' class in green, 'w/G swi+++' class in yellow. The rotation axis (red dot) and the rotation angles with respect to the 'w/oG swi-' class are indicated at the bottom.

  an intermediate swivelled conformation without a protein regulator or DNA signal. It suggests that swivelling could simply be the result of intrinsic conformational flexibility of RNAP.The remaining three classes ('w/G swi-' class, 'w/G swi+' class, 'w/G swi+++' class) have strong density for NusG, thus they correspond to the NusG-EC. Similar to RNAP without NusG, these classes (60% of all particles) displayed a range of swivelling angles from 0.9° to 3.3° (Figure31), which suggests that RNAP can also access a continuum of swivel module positions in presence of NusG. Similar results were independently obtained from 3D classification in Relion.

Figure 30

 30 Figure 30 Conformational change in classes lacking NusG. Classes lacking NusG ('w/oG swi-', 'w/oG swi++') were aligned based on the RNAP core module. A reconstruction from the nonswivelled class ('w/oG swi-' ) contains stronger density for the upstream DNA (top image on the left; in this view, downstream DNA is sandwiched between the β and β' subunit). On the bottom left the structure is rotated 90° with respect to the top image. The SI3 domain is closer to the β-lobe in a class representing the more swivelled compared to the class representing the nonswivelled state, respectively (compare 'w/oG swi-' in pink to 'w/oG swi++' in blue). The image on the right shows only the swivel module of structures refined against both classes and aligned based on the RNAP core module. The extent of swivel module rotation between the two classes is about 2.4°. The red circle designates the rotation axis. The yellow dashed line and pink-orange line indicate the swivel module positions.

Figure 31

 31 Figure 31 Superposition of NusG-EC classes that contain NusG. Structures refined against reconstructions of the three classes ('w/G swi-', grey; 'w/G swi+', lavender; and 'w/G swi+++', blue) were aligned based on the RNAP core module. Their swivel modules display different extents of rotation: least swivelled ('w/G swi-' class), intermediate ('w/G swi+' class), and more swivelled ('w/G swi+++' class).

Figure 32

 32 Figure 32 Comparison of nt-DNA and upstream DNA density between classes with or without NusG. The structure is coloured by chain and labelled next to the density maps; protein backbone is shown as ribbons and DNA as sticks. RNAP is in blue, nt-DNA and t-DNA are in lavender and violet; RNA is in green and NusG in red. The reconstruction on the left ('w/G swi-') is in the non-swiveled state and contains NusG. It has better defined upstream DNA and nt-DNA densities compared to reconstructions lacking NusG ('w/oG swi-', and 'w/oG swi++'). The same is true for reconstructions containing NusG but with increased swivel angles ('w/G swi+', and 'w/G swi+++'; data not shown).

Figure 33

 33 Figure 33 Electrostatic potential of NusG-EC surface. The fours figures are all NusG-EC in the same view, the top left structure illustrates electrostatic surface, the right bottom structure illustrates the cartoon form. The four structures show a gradual transition of electrostatic surface form to cartoon form. Positively charged regions are in blue and negatively charged regions in red. NusG is positioned on the right top corner, between β-clamp and β-protrusion. the modelled nucleic acids and NusG as a cartoon in purple and in green. NusG and RNAP create a positively charged environment for the upstream DNA.

Figure 34

 34 Figure 34 The flexible CTD domain of NusG.

Figure 35

 35 Figure 35 The upstream DNA is enclosed by NusG and RNAP. The RNAP is shown as a surface in blue. The DNA is shown as a cartoon (violet). NusG is shown in both surface and as a ribbon (red). The black dashed circle region indicates that upstream DNA is enclosed by NusG and RNAP, the dark blue (RNAP) and dark red (NusG) are the regions closest to the DNA (less than 10 Å to upstream DNA).

Figure 36

 36 Figure 36 β-protrusion of RNAP moves away when NusG binds to the EC. A structural superposition of the complex is shown based on the RNAP core module (RNAP in non-swivelled conformation, yellow surface; RNAP in swivelled conformation, shades of blue and NusG in red) in. The β-protrusion (in dark blue) moves further away from NusG, while the β-lobe moves closer to the SI3 domain as a result of swivelling. On the bottom, right corner, four additional contacts are shown when RNAP adopts nonswivelled state (β-Y62, β-A380, β-Y367, β-G373).

Figure 37

 37 Figure 37 Overview of consensus refinement of the NusA-EC complex. A) A cryo-EM density map for the NusA-EC with a nominal resolution of 4.1 Å (with a transparent map on the left). The structure (shown as backbone ribbon) is coloured by chain and labelled next to the density map. RNAP subunits are in different shades of blue, nt-DNA and t-DNA are in lavender and violet; RNA is in green and NusA in yellow. On the right, the locally sharpened EM map is shown. B) A zoom-in at the active centre of RNAP, showing the DNA-RNA hybrid in the post-translocated state. C) Angular distribution plot shows random particle orientation of the NusA-EC reconstruction. D) Fourier shell correlation (FSC) plot for half-maps of the NusA-EC reconstruction estimates the nominal resolution to be 4.1Å using the gold-standard Fourier shell correlation criterion.

Figure 38

 38 Figure 38 Structural heterogeneity of NusA. Map superposition based on structural superpositions of RNAP of several reconstructions following 3D classification shows NusA in different orientations relative to RNAP, which reflects its flexibility. NusA can rotate or translate in several directions. NusA in violet is in the consensus conformation used as a reference for comparison.

  The non-swivelled reference structure ('w/oG swi-', see previous section on NusG-EC) is in yellow and the consensus NusA-EC structure is in violet (small inset, top left). The maximally swivelled structure is coloured in blue (centre). The red circle indicates the axis of rotation. The yellow dashed line and solid violet line show the position of the swivel modules in the reference and maximally swivelled state with the swivelling angle of 5.5° indicated.

Figure 41

 41 Figure 41 The swivelled conformation of the NusA-EC.

Figure 42

 42 Figure 42 Comparison of RNA-DNA hybrid between swivelled and non-swivelled state in the NusA-EC.

Figure 43

 43 Figure 43 Comparison of nt-DNA and upstream DNA between NusA-EC and NusG-EC. Maps and atomic models of NusG-EC (blue) and NusA-EC (pink) are shown superposed on the left and each of them separately on the right. Protein backbones are shown as ribbons and nucleic acids as sticks. The labels with arrows on the right highlight different elements of the NusA-EC or NusG-EC. In NusG-EC, nucleic acids are coloured dark blue, NusG and RNAP light blue; while in NusA-EC nucleic acids are dark pink, NusA and RNAP are light pink. The upstream DNA and nt-DNA are better defined in NusG-EC compared to NusA-EC.

  It is consistent with the results from 3D classification, because the class corresponding to a swivelled RNAP, refined to a higher swivel angle compared to the swivelled NusG-EC. Finally, it agrees with the biochemical role of NusA, which favours and stabilizes paused states of RNAP and a swivelled state is likely a common intermediate for them. NusA binds the RNA coming from the RNA exit channel and indirectly the RNAP zinc-finger thereby favouring the swivelled conformation.

Figure 44

 44 Figure 44 RNA at the RNA exit channel in the swivelled and nonswivelled state. A) NusA and the RNAP zinc finger (ZF) are shown as surface coloured by their electrostatic potential on the left and shown as cartoon on the right. The positively charged regions are in blue and negatively charged regions in red. NusA is coloured in yellow, RNAP in grey and the ZF of RNAP in blue. The S1 domain of NusA; β-flap domain, ZF and ω-subunit of RNAP are labelled. B) The cryo-EM density maps and the corresponding models are coloured by components: RNAP in blue and NusA in yellow. RNA (density in green) is not modelled. The density for RNA appears and the density for NusA is better resolved when RNAP adopts the swivelled conformation compared to the non-swivelled conformation (compare panel B and C).

Figure 45

 45 Figure 45 Overview of consensus refinement of the NusA-NusG-EC. A) A cryo-EM density map for the NusA-NusG-EC with a nominal resolution of 4.2 Å (with a transparent map on the left). The structure (shown as backbone ribbon) is coloured by chain and labelled next to the density map. RNAP subunits are in different shades of blue, nt-DNA and t-DNA are in lavender and violet; RNA is in green and NusA in yellow. On the right, the locally sharpened EM map is shown. B) A zoom-in at the active centre of RNAP, showing the DNA-RNA hybrid in the post-translocated state. C) Angular distribution plot shows random particle orientation of the NusA-NusG-EC reconstruction. D) Fourier shell correlation (FSC) plot for half-maps of the NusA-NusG-EC reconstruction estimates the nominal resolution to be 4.2Å using the gold-standard Fourier shell correlation criterion.

Figure 46

 46 Figure 46 Heterogeneous refinement of NusA-NusG-EC in CryoSPARC. RNAP subunits are coloured in different shades of blue; nt-DNA and t-DNA are in lavender and violet; RNA in green, NusA in yellow and NusG in red. The consensus refinement from all particles in the dataset (on top) has the highest resolution. The heterogeneous refinement separated two major classes: NusG-EC and NusA-NusG-EC. NusG-EC class showed very weak density for NusA. The density for NusA in NusA-NusG-EC on the bottom right is shown in yellow and at a lower contour level.

Figure 47

 47 Figure 47 Swivelling angle of NusA-NusG-EC.

Figure 48

 48 Figure 48 Density of NusG becomes weaker when RNAP swivels at high angle. RNAP is coloured in blue, nt-DNA and t-DNA in lavender and violet; NusA in yellow and NusG in red. The region where NusG binds to RNAP is indicated by a white dashed circle.

  exponential term was insufficient to model the data accurately. Both RNAP species ('fast' and 'slow') can be followed by quantifying the extension of RNA U29 to G31 as a function of time (Figure49 B).Therefore, the sum of RNAPslow and RNAPfast represents all the RNAPs in the ensemble (Figure49 C). Modelling the experimental data (Figure 49 D) suggests roughly onethird of the population to be RNAPfast (38%) and two-thirds to be RNAPslow (62%) at the his-pause site, indicating two distinct RNAP species in absence of any TFs. When adding NusA, I observed a general decrease of RNAP pause-escape rates. Both RNAPfast and RNAPslow species were affected and the escape rates decreased roughly 2-fold. In addition, the fraction of RNAPslow in the ensemble increases to fourfifths (84%, Figure 49 C, 1 D, compare yellow vs. black curve, and pause half-lives). In contrast to NusA, addition of NusG causes a general increase of RNAP pause-escape rates. Both RNAPfast and RNAPslow species show increased pause-escape rates, and the fraction of RNAPfast in the ensemble increases slightly (43%) (Figure 49 C, 1 D, compare red vs. black curve, and pause half-lives). As shown in the NusA-NusG-EC structure, the CTD of NusG competes with NusA for the RNAP FTH, which is an important binding site for NusA. I speculated whether these two factors can cancel each other's effect, and whether the CTD of NusG plays a role in this competition. Therefore I mixed the RNAP, NusA and with either NusG-FL or NusG-NTD. When both NusA and NusG (NusG-FL or NusG-CTD) were presented simultaneously in the reaction, the accumulative results (RNAPfast + RNAPslow) were indistinguishable from each other and were similar to RNAP alone, yet with slightly longer pause duration (Figure 49 C, 1 D, compare green and purple vs. black curve, and pause half-lives). This observation indicates that NusA may have a stronger effect than NusG. These results indicate that NusA and NusG have opposing regulatory effects on RNAP at the his-pause site. NusG cannot fully compensate NusA's effect, even when present in excess over NusA. This suggests that NusA might be the dominant regulator in the RNA-hairpin induced pause. (Figure 49). The kinetic parameters show that the two domains of NusG seem to play different roles during pausing. For instance, NusG-NTD alone is sufficient to oppose NusA's effect.

Figure 49

 49 Figure 49 Combined effect of NusG and NusA on his-pause. A) A schematic of the nucleic acid scaffold used in the assay (RNA in blue-green, t-DNA in purple and nt-DNA in lavender). A hairpin forms in the nascent RNA, which stabilizes a paused state of RNAP at U29. Upon addition of rGTP RNAP escapes from the pause and incorporates two nucleotides (RNA product at G31). B) A representative gel shows the time course of pause escape. RNAP pauses at U29 or escapes from the pause and elongates until G31. The RNA substrate was labelled with 32P-γ-rATP at the 5'-end, aliquots of were taken from the reaction at different time points (indicated on top of the gel) and separated on a 15% denaturing polyacrylamide gel. C) The assay was performed with different combinations of transcription factors: RNAP alone; RNAP with NusA; RNAP with NusA and NusG; RNAP with NusA and NusG-NTD; RNAP with NusG. Each experiment was repeated three times, and paused products were quantified and plotted as shown in the graph. NusA enhances the his-pause and NusG decreases pausing. NusG or NusG-NTD in combination with NusA makes RNAP pause longer compared to RNAP without transcription factors but shorter than RNAP with NusA alone. D) The kinetic parameters were obtained from fitting the experimental data in (C) using the equation shown above.

  Figure50C-E, compare sum of RNAPfast, and RNAPslow) and escapes from the ops-pause as two kinetic species (0.13 sec-1 and 0.02 sec-1, respectively). The addition of NusG did not dramatically change the fraction of RNAP entering the pause (30%, Figure50 E, compare the sum of RNAPfast and RNAPslow). Instead, NusG mostly affects RNAPslow and increases its escape rate to unify them into one kinetic species (please note that the fitting was done under the assumption of two kinetic species to be consistent but both species end up to have virtually identical escape rates). This single species, although having a slightly slower transcription rate than RNAPfast without NusG (0.09 sec-1 vs. 0.13 sec-1) overall escapes faster and thus, paused RNAP has a shorter half-life in presence of NusG (Figure50 D, 2E, compare red vs. grey curve, and pause half-lives).On the contrary, the presence of NusA extends pausing. First, a larger fraction of RNAP enters the pause (65%, Figure50 E, compare the sum of RNAPfast and RNAPslow). This increase in paused RNAP mostly occurs because the fraction of RNAPfast doubles (48% in presence of NusA vs.

Figure 50

 50 Figure 50 Combined effect of NusG and NusA on the ops pause.A) DNA template used in transcription assay. A linear template DNA was generated by PCR amplification from the vector containing T7A1 promoter (transcription start site indicated by bent arrow), U-less region ending with A48, ops-pause site, and transcription terminator (release site at +217). B) A representative gel showing all the RNA products of transcription in the absence of TFs. Each column of the 10% polyacrylamide urea gel indicates the transcripts generated from the template at different time points: 10'', 20'', 30'', 1', 1'30'', 2', 3', 5', 10', chase. At time point 0, the halted A48 elongation complex was formed. Elongation was restarted upon addition of all the rNTPs. RNAP pauses at ops-pause site (U60). Positions of the halted (A48) and paused (ops U60) EC are indicated by arrows on the left. C) Enlarged part of the gel at the pause site. RNA products at U60 in the absence or presence of NusG and/or NusA are indicated on the left side of the gel. D) Quantification of paused RNA at U60 over time. NusG suppresses ops-pause, NusA enhances it, while NusG and NusA in combination do not seem to have an effect on pausing anymore. E) The kinetic parameters were obtained from fitting the experimental data in D) using the equation shown above.

Figure 51

 51 Figure 51 Combined effect of NusG and NusA on the ρ-dependent termination. A) DNA template was generated by PCR amplification. It contains a T7A1 promoter (transcription start site indicated by bent arrow), a U-less region ending with A29, the phage lambda cro gene, a binding site for ρ (rutA, boxB and rutB), followed by the termination region (early and late). (release site at +389) B) A representative gel shows terminated transcription products in presence of different combinations of TFs (indicated on the top of the gel). The early and late termination sites and run-off region, as well as the size of the DNA marker, are indicated next to the gel.

Figure 52

 52 Figure 52 Quantification of RNA products at termination sites and run-off region. The overall termination efficiency (ratio of terminated species over terminated plus run-off) has been determined for different combinations of transcription factors (indicated below the plot).

Figure 54

 54 Figure 54 Transcription assay to monitor ρ-dependent termination kinetics in presence of NusG and/or NusA.A) Different combinations of TFs (indicated on the top of the gel) are added to the reactions. The DNA marker labelled on the left was used for approximation of the size of RNA products. The terminators and run-off regions are represented by bars on the right side of the gel. To follow the reaction, samples are taken at different time points as indicated on top of the gel. In the absence of TFs, most RNA products bypass terminators and transcription ended in the run-off region (transcripts could be even longer than the total length of DNA template, release site at +389). ρ made RNAP terminate transcription at the late terminator. NusA increased pausing at the terminators but RNAP eventually extended those transcripts over time even in presence of ρ. Termination occurs earlier in presence of NusG that increased the termination efficiency. Termination efficiency increased even more when both NusG and NusA are present in combination with ρ at the early termination site. B) Negative control (without ρ) of ρ-dependent termination assay over time shows no significant differences except NusA may stimulate slightly more pausing in the early termination region. C) Mathematic model used to simulate RNAP arriving at the early termination region of a ρ-dependent terminator over time. The curves represent all RNAPs terminate transcription at the early termination site (a); all RNAP by-pass the early termination site (b); some fraction of RNAPs terminate and some by-pass (80% in c and 30% in d). D) A linear combination of models representing RNAP bypassing and RNAP terminating is used to model the experimental data. Parameters R: the amount of RNAP at the termination site; t: time; σ: the width of the gaussian peak accounting for RNAP desynchronization before reaching the terminator; time t0: (=11.75 s) average arrival time at terminator, which was determined by trial and error to yield the smallest errors on the amplitudes; Poff: the probability of RNAP to fall off from the DNA in the terminator region and release the transcript; 𝜏pause: the pause time; A: represents the probability of pausing in the termination region; R0: accounts for background signal. E) Quantification of the termination efficiency in presence of NusG and/or NusA over time. RNA products were quantified as triplicates and plotted at different time points at the early terminator. The reaction curves with different TFs were superimposed, RNAP + ρ, RNAP + ρ + NusA, RNAP + ρ + NusG and RNAP + ρ + NusA + NusG are coloured differently: dark blue, orange, grey, yellow and light blue, respectively. F) Table of parameter values obtained for each reaction from fitting the model to the experimental data. Error estimates are in parentheses (nd*: not determined, here indicates termination) E) The negative control (NusG-NTD) shows that deletion of NusG-CTD abolishes the effect of NusG on ρ-dependent termination, consistent with a model, that implies NusG-CTD in the stimulatory activity on ρ (the

  RNAP and is not necessarily induced by a pause signal in the DNA. RNAP can oscillate between the swivelled and non-swivelled conformations randomly. The swivelled conformation likely facilitates entry into the elemental pause state, which serves as a precursor for long-lived pauses. This swivelling movement (the rotation of clamp and shelf) is accompanied by additional observations: it prevents trigger loop folding (the trigger-loop connected SI3 domain cannot change its position); it reduces the angle between upstream and downstream DNA; it loosens contacts between RNAP and the RNA-DNA hybrid and possibly destabilizes the hybrid (this may slow down the rate of RNA extension); it widens the RNA exit channel (and consequently allows formation of RNA secondary structures within the exit channel); it facilitates upstream movement of the lid loop (an element that stacks on the upstream end of the RNA hybrid) to a position away from the RNA-DNA hybrid (may allow RNAP backtracking by allowing reannealing of upstream t-DNA with RNA) and kinking of the bridge helix. According to my observation, the BH is kinked in the active site when RNAP is swivelled (although less kinked than in the NusA-his-PEC complex, which adopts a highly swivelled state). A highly kinked BH might prevent the template base and rNTP from entering into the active site and thus prevent nucleotide addition.

  favouring the swivelled conformation of RNAP. Consistent with this proposal, the NusA-EC is able to swivel to higher angles RNAP than the NusG-EC or the NusA-NusG-EC. I propose that NusA favours the swivelled conformation by pulling on the ZF, which is a part of the rigid swivel module. The interaction between ZF and NusA appears to be indirect and mediated by the nascent RNA. Similar interaction between ZF and NusA have been recently observed in ρ-dependent termination, where NusA cooperates with the ZF to block ρ in 'open-ring' conformation[START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF]. I propose that the NusA -ZF interaction could be a driving force for the conformational change and for the shift of the conformational equilibrium towards the swivelled state.I propose that NusA does not interact with the ZF and with the RNAP omega subunit at the same time. Instead, I suggest that RNAP oscillates between the swivelled and non-swivelled conformation and NusA interacts either with the ZF or with the omega subunit respectively. This hypothesis is consistent with a recent study[START_REF] Said | Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ[END_REF], which indicated that deleting omega subunit potentiated ρ-dependent termination. Termination likely requires a swivelled conformation (and RNAP in Said et al., appears to be in a similar swivelled conformation compared to my structure) -according to my hypothesis, deleting omega should favour the swivelled state and thus favour termination.

  conformation. The ECs reported in my studies all adopt both swivelled and non-swivelled conformation, but they all contain RNA-DNA hybrids in the post-translocated state. My work shows that the swivelled conformation does not necessarily correlate with the half-translocated RNA-DNA hybrid. The half-translocated state might only happen when RNAP encounters a pause inducing sequence such as the E. coli hispause. Additional elements such as an RNA hairpin in the nascent transcript may further stabilize this state.

  According to my observation, I propose that during transcription elongation NusG suppresses pausing mostly by shifting the conformational equilibrium of RNAP to a non-swivelled state irrespective of a pause signal. Additionally, NusG also stabilizes nt-DNA as well as upstream DNA by extending a positively charged surface from RNAP. Stabilization of the upstream DNA might favour forward

  the 'fast' and 'slow' species into one single species. NusA and NusG partially cancel each other's effect in the case of the E. coli his-pause or fully cancel their individual effects in the case of the ops-pause as was shown in my kinetic assays. NusG-NTD alone has the same accumulative effect as NusG-FL on reducing the pausing enhancement of NusA on the his-pause. This indicates that the competition between NusA and NusG in the RNA hairpin stabilized pausing is not coming from the competition for FTH binding because NusG-CTD was absent. One possible explanation is that NusA, by interacting with the RNA hairpin in the RNA exit channel and with ZF favour the swivelling conformation of RNAP. Yet, NusG might tune RNAP into non-swivelled conformation, which might cause a partial reduction of pausing.

  summarize, I propose a series of regulatory events at the early terminator: 1) RNAP recognizes the pausing signal; 2) NusA occupies the FTH, increases the probability of RNAP pausing and stabilizes RNAP in the paused conformation; 3) ρ might be able to interact with ZF and transform EC into termination. Otherwise, the free NusG-CTD might also activate ρ and redirect all the paused RNAP into termination. All these steps together complete the transcription by termination.VII. GLOBAL PICTURE IN PRESENCE OF NUSA, NUSG, RIBOSOME, AND RHOAfter transcription-initiation and promoter-escap, both NusA and NusG approach and bind to RNAP. They influence the equilibrium between swivelled and non-swivelled state to control RNAP's pausing or its escape from pausing. Once the lead ribosome catches up with RNAP, NusG-CTD could bind the ribosome to couple translation with transcription. This may have the effect of stabilizing NusG on the RNAP EC and thus to prevent pausing further and increase the rate of transcription. When RNAP is far ahead of the ribosome, NusA can increase pausing to slow down RNAP. When RNAP reaches the termination sites, NusA occupies the FTH of RNAP and increases the fraction of paused RNAP. The free CTD of NusG activates ρ and redirects all paused RNA species to termination.

  during my four-year PhD training, I obtained three cryo-EM structures of RNA polymerase elongation complexes (RNAP EC): E. coli RNAP EC bound to NusG, E. coli RNAP EC bound to NusA, and E. coli RNAP EC bound to both factors simultaneously. This is the first time that I could resolve full length NusG bound to an E. coli RNAP EC in a cryo-EM reconstruction. Previous biochemical studies demonstrated that NusA and NusG have antagonistic effects on the transcription rate. My study provides structural and mechanistic details on this aspect: i) both NusG-CTD and NusA-NTD interact with the same RNAP module, namely the RNAP flap-tip-helix (FTH); ii) both NusA and NusG influence the swivelling equilibrium of RNAP but shift it in the opposite direction. Therefore, I propose this might be the mechanism of tuning RNAP before encountering a pause signal. However, during ρ-dependent termination, NusA and NusG seem to play different roles and contribute to efficient early termination. Furthermore, NusG is able to physically couple the transcription and translation machineries by bridging RNAP and ribosome. We proposed that this would align the nascent transcript with the ribosomal helicase to maintain a single stranded conformation and thus avoid the formation of inhibitory secondary structures in the transcript. Furthermore, as long as ribosome and RNAP are in close spatial proximity and share the mRNA, NusG occupancy is likely maintained due to avidity effects (NusG-CTD binds the ribosome, NusG-NTD binds RNAP) and this in turn should increase transcription rates and stability of expressome. Finally, once the ribosome has encountered a stop codon, the NusG-CTD is free to assist and ensure timely ρ-mediated transcription termination. RNAPs from different organisms share common basic mechanism of transcription elongation, and therefore, our findings on transcription regulation may be applicable to all multi-subunit RNAPs. However, in B. subtilis, NusG shows pausing increment regulation instead of decrease in E. coli. It would be interesting to study such mechanism, which might give new insights. Furthermore, it would be important and indispensable to answer the following questions for better understanding on transcription regulation: How does the structure of a RNAP EC with both factors simultaneously bound look like on a pause site? Does NusG stimulate ρ factor to adopt a closed-ring conformation or is it involved in recruiting ρ to the EC? If that is the case, what are the mechanisms?

  . In 2018, Guo et al. obtained a single particle cryo-EM reconstruction of NusA bound to an EC paused at the his-pause

	which may aid in RNA folding and accommodates the nascent RNA
	hairpin. RNAP recognizes the pause signal and enters an elemental
	pause. The S1-KH1-KH2 motif of NusA and zinc finger motif of RNAP
	create a positively charged surface, which may guide the transcript
	away from the exit channel in agreement with crystal structures of
	NusA bound to RNA (Hein et al., 2014; Said et al., 2017). The RNA-DNA
	hybrid has been observed in a half-translocated state (Guo et al., 2018;
	Kang et al., 2018b) (RNA fully translocated into post-translocated state,
	t-DNA not yet fully translocated) which inhibits the rNTP binding
	because the transcribed nt-DNA base is still in the active site and
	including a nascent RNA hairpin at a 3.7 Å resolution. It suggested how
	RNAP becomes inactive so the elongation process is slowed down at the
	his-pause site. The NusA AR2 binds to the α1-CTD of RNAP, releasing

autoinhibition of the S1, KH1, and KH2. NusA-NTD interacts with the FTH of RNAP, pulling it away from the RNA exit channel and, together with the S1 domain, provides an extended positively charged surface, modifies RNAP into a long-lived paused state. Recent research found that pausing efficiency can be increased by expanding the interface between the RNA and NusA. Thus, it has been suggested that NusA enhances pausing 3-fold by stabilizing the RNA structure

Table of

 of

	parameter values obtained for each reaction from fitting the
	model to the experimental data. Error estimates are in parentheses (nd*: not
	determined, here indicates termination) E) The negative control (NusG-NTD) shows that
	deletion of NusG-CTD abolishes the effect of NusG on ρ-dependent termination,
	consistent with a model, that implies NusG-CTD in the stimulatory activity on ρ (the

transcription profile looks similar to a reaction with 'RNAP + ρ + NusA' without NusG; compare panel A).

III. RESULTS -TABLES3

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II. RESULTS -TABLE

 S2

RESULTS

SUPPLEMENTARY DOCUMENTS

Résumé en français suivi des mots-clés en français L'ARN-polymérase (ARNP) est l'enzyme clé de la transcription. Elle est étroitement régulée par des facteurs au cours du cycle de la transcription. Les deux facteurs NusA et NusG, ont un effet opposé sur la pause transcriptionnelle. Le but de ma thèse est d'utiliser la cryo-EM en analyse de particules isolées combinée à des analyses biochimiques pour mettre en évidence la régulation de l'ARNP par NusA et NusG, et ces deux facteurs ensemble. Nous démontrons que l'ARNP elle-même a un mouvement dynamique constant entre conformation non-pivotante et pivotante. NusA stabilise l'ARNP dans une conformation pivotante qui est proche de l'état de pause, alors que NusG favorise un état non-pivotant de l'ARNP. NusG-CTD est en compétition avec NusA sur un même site de liaison, le module Flap-Tip-Helix de l'ARNP. Les résultats biochimiques ont montré que NusA et NusG, compensent leurs effets et modulent la vitesse de transcription. Cependant, dans le contexte de la terminaison, rho-dépendante, NusA et NusG, ensemble pourraient augmenter l'efficacité de la terminaison précoce.

Mots clés :

L'élongation & la terminaison de la transcription, NusG & NusA, Cryo-EM, la dynamique de l'ARNP

Résumé en anglais suivi des mots-clés en anglais

The RNAP (RNA polymerase) is the key enzyme in transcription. RNAP is tightly regulated by factors during transcriptional cycle. Two speed control transcriptional factors (TF) NusA and NusG have the opposite effect on transcriptional pausing. The aim of my work is to use single particle Cryo-EM combine with biochemistry analysis to bring out the regulation of NusA and NusG, and both TFs together on the RNAP. We demonstrate here that RNAP itsself has a constant dynamic movement -non-swiveled to swiveled conformation. NusA stabilized the RNAP to a swiveled conformation which close to the paused state, however NusG enhance the RNAP to an non-swiveled state. NusG-CTD compete with NusA on a same binding site, the Flap-Tip-Helix (FTH) module of RNAP. The biochemistry results showed that these two FT NusA and NusG, compensate the effect of each other and modulate the transcriptional rate in different transcriptional pausing context (class I and class II pausing). However at the termination rho-dependent context, NusA and NusG together could increase the termination efficiency at the terminator I site.