Keywords: Staphylococcus aureus, ribosome, translation initiation, cryo-EM Cryo-electron microscopy study of translation initiation complexes from Staphylococcus aureus

Dr. Celia Plisson-Chastang and Dr. Gulnara Yusupova who agreed to evaluate my PhD work. I am very grateful to meet them in Strasbourg and to discuss.

I am grateful to my PhD supervisor Yaser Hashem for giving me a chance to work in his team, for all our discussions, for his help and motivation. Every day I learned a lot while working in his laboratory. Also, I met so many wonderful people in Strasbourg and in Bordeaux. It was a pleasure to work in your lab.

I am thankful to my co-supervisor Marat Yusupov for his support and patience. Time spent in his laboratory was productive and useful for me.

I express my gratitude to Stefano and Angelita for sharing their experience with me, for helping me with my main project and for all our discussions. Special thanks to Pascale Romby for all our discussions that gave me a fresh view on my project.

I would like to thank everyone in all three labs I worked in, starting from Iskander and Simone for teaching me and helping me with my projects. Then, to Jay, Quentin and Anthony for all our useful discussions and your contribution in my main project, to Clair and Adeline for technical support. Thanks to people from my second lab: Alexey, Nemanja, Sasha, Justine, Mélanie and Lasse for all their useful advices and to Irina, Salvatore, Olga, Yury and Mumin for their constant support. Also, thanks to people from my third lab in Bordeaux: Marie, Camila, Stéphanie, Florian, Aline, Mayara, Ewelina for their support during my last year of PhD. Special thanks to Anthony and Florent for your help in the last part of my PhD and to Heddy for grids preparations, data acquisition and image processing and also our useful discussions. Without all these people it would be much more difficult to do my projects.

Thanks to Pascale Romby's (Isabelle, Anne-Catherine, Emma, Laura and Lucas) and Axel Innis (Alba, Mélanie, Elodie, Natacha, Mecit, Aitor and Anne-Xander) laboratories for the opportunity to sometimes work in their labs.

Danièle and Léandra for the incredible support and help with all my documents.

Johana, Lauriane and Philippe for the mass spec analysis of my samples. Without their commitment and effort this work would not be possible.

Many thanks to my friends Irina, Lyuba, Elena, Fatima, Salvatore, Olga and Yury for your support and incredible time spent together.

TABLE OF FIGURES AND TABLES

Figure 1. The small ribosomal subunit with tRNA binding sites and decoding center Figure 2. The large subunit major functional sites are the tRNA binding sites, the peptide exit tunnel that extends through the body of the large subunit and the peptidyl transferase center. Antibiotics used against S. aureus and the mechanism of their resistance 40 Table 5.

List of ribosomal proteins found in S. aureus ribosome sample by LC-MS/MS analysis 49 Table 6.

List of non-ribosomal proteins found in S. aureus ribosome sample by LC-MS/MS analysis 51 Table 7.

Characteristics of microscopes used for data acquisition 73 Table 8.

List of proteins found in S. aureus 30S ribosome sample by LC-MS/MS analysis 81 Table 9.

Variations of prepared complexes collected on Talos microscope and processed CryoSparc software 92 Table 10. List of proteins found in C. albicans 80S ribosome sample by LC-MS/MS analysis Amino acids are described according to the standard nomenclature with three letters. All ribosomal proteins named according to the current nomenclature [START_REF] Ban | A new system for naming ribosomal proteins[END_REF].

INTRODUCTION

Protein synthesis, also called mRNA translation, is a conserved process that is carried out by the ribosome in all living cells. In 2009, Konstantin Bokov and Sergey Steinberg, based on the 3D-structure of the ribosomal RNA from Escherichia coli, suggested that ribosome could be formed as a result of a gradual evolution from a very simple small RNA molecule (proto-ribosome). Each new ribosome insert was sequentially added, making the ribosome more stable [START_REF] Bokov | A hierarchical model for evolution of 23S ribosomal RNA[END_REF].

Core of the ribosome

The ribosome is an important machine and is a major link between genes and proteins.

It has an asymmetric structure with a common core between prokaryotic and eukaryotic organisms. Structurally and functionally, the ribosome includes a large and a small subunits that are hybrid molecules made of ribonucleic acid (ribosomal RNA) and proteins. The average ratio between rRNA(s) and proteins is 2:1 in bacteria and about 1:1 in eukaryotes. In the mitochondrial and chloroplast ribosomes ratios are 1:2 and 3:2 respectively (Sharma and [START_REF] Agrawal | Structural aspects of mitochondrial translational apparatus[END_REF].

Small ribosomal subunits (SSU) from prokaryotes (30S) and eukaryotes (40S) have similar shapes, and some of their landmarks as dubbed in anthropomorphic terms such as 'head', 'body', 'beak' and 'shoulder'. They are responsible for selection of the aminoacyl-tRNA (aa-tRNA) according to the mRNA sequence and have a functional mRNA and tRNA binding sites: aminoacyl-tRNA (A-site), peptidyl-tRNA (P-site) and the exit site for the

The Ribosome deacylated-tRNA (E-site) (Fig. 1). During translation, tRNAs are translocated from the A-to the P-site and from the P-to the E-site. E), the peptide exit tunnel that extends through the body of the large subunit, and the peptidyl transferase center (PTC) [START_REF] Melnikov | One core, two shells: bacterial and eukaryotic ribosomes[END_REF].

Features of prokaryotic and eukaryotic ribosomes

Despite significant similarities in the structure of ribosomes between prokaryotes and eukaryotes, there are many peculiarities in structures and regulation mechanisms of ribosomes from different organisms and also in the process of protein synthesis in general. The prokaryotic ribosome is formed by some 54 ribosomal proteins and 3 ribosomal RNAs (16S, 23S and 5S), while the eukaryotic ribosome is composed of some 79 ribosomal proteins and 4 ribosomal RNAs (18S, 25S, 5.8S and 5S). The fully assembled prokaryotic ribosome has a sedimentation coefficient (particle deposition rate during ultracentrifugation) of 70S, its LSU of 50S and SSU of 30S, whereas the eukaryotic ribosome is an 80S because of its ~40% molecular weight, consisting of an LSU of 60S and an SSU of 40S (Fig. 3). The archaeal ribosome have a size and composition similar to bacterial ribosomes: they contain three rRNA molecules (16S, 23S and 5S) and 50-70 proteins depending on the species. However, the primary structures of archaeal rRNA and r-proteins are closer to eukaryotes (Table 1).

Bacterial Yeast Human

Figure 3. Composition of bacterial and eukaryotic ribosomes: RNA in light grey, proteins of small ribosomal subunit in blue and proteins of large ribosomal subunit in red. The figures are based on X-ray and Cryo-EM structures from [START_REF] Jenner | Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis[END_REF], Ben-Shem et al., 2010, Armache et al., 2010.

Table 1. Composition and characteristics of ribosome from different species (Yusupov et el., 2001[START_REF] Spahn | Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes: The IRES Functions as an RNA-Based Translation Factor[END_REF][START_REF] Schuwirth | Structures of the bacterial ribosome at 3.5 A resolution[END_REF], Ben-Shem et al., 2010[START_REF] Armache | Promiscuous behaviour of archaeal ribosomal proteins: implications for eukaryotic ribosome evolution[END_REF]. Briefly, the 80S ribosome presents many features that are not found in the bacterial ribosomes. For instance, additional rRNA helices (also called expansion segments), additional ribosomal proteins and protein extensions organized in tails.

Eukaryotic-specific ribosomal proteins and extensions

In the eukaryotic ribosome, specific proteins for the small ribosomal subunit are S1, S4, S6, S8, S10, S12, S17, S19 -S31 (Fig. 4). For the large ribosomal subunit specific proteins are L6, L8, L13, L14, L15, L18, L19 -L22, L24, L27, L30, L31 -L43 (Table 2, Fig. 5).

There are a group of proteins with unusual folds long tails and extending loops (S3, L4, L22, L23 and L29). With the determination of ribosome structures from different organisms, it became clear that these unusual protein folds are even more abundant in eukaryotes than in bacteria. The function of these insertions and extensions is to interact with other eukaryoticspecific proteins and rRNA extension segments, which may facilitate the assembly of eukaryotic-specific components.

RNA expansion segments (ES)

Expansion segments (ESs) can be defined as the additional rRNA insertions the eukaryotic ribosome compared to their bacterial counterpart. They can be found on both the SSU (labeled with an "S") and the LSU (labeled with an "L"), accordingly. Although it has been shown that ES act as platforms for the binding of proteins that modulate translation across evolution (Armache et al., 2010, Fujii et al., 2018, Fig. 6 and7). Protein synthesis can be divided into 4 main steps: initiation, elongation, termination and recycling.

Initiation

The initiation phase involves the formation of a first complex on the small ribosomal subunit that sets the position of the initiation codon of mRNA (AUG), as well as the initiator tRNA (fMet-tRNA fMet ) at the SSU P-site. Bacterial translation initiation will be covered in the details later on during this introduction and will be briefly mentioned in this section.

In bacteria, this process is mainly controlled by three translation initiation factors: IF1, In eukaryotes, the initiation process is more complex and includes a dozen of eukaryotic initiation factors. It begins with the formation of the preinitiation complex (43S). Firstly, the ternary complex (TC) is formed from eukaryotic initiation factor 2 (eIF2), guanosine triphosphate (GTP) molecule and initiator tRNA (MettRNA Met i). Subsequently, this complex binds to the post-recycled complex that consist of small ribosomal subunit, eukaryotic initiation factors 1, 1A and 3 (eIF1, eIF1A, eIF3) and the ATP binding cassette E1 (ABCE1).

Protein synthesis

Then the mRNA is recruited and after a series of large-scale conformational rearrangements the 48S complex is formed and scan for the start codon. After start codon recognition, almost of the initiation factors disassemble in sequentially and eukaryotic initiation factor (eIF5B) marks the last steps of the process with its binding to the GTPase center of the SSU, which stimulates the association of both ribosomal subunits, prior to it is released before the elongation process commences (from review Guca and Hashem, 2018, Fig. 9).

Elongation

The elongation process is better conserved compared to initiation among all species. It starts with the delivery of a new aminoacyl-tRNA (aa-tRNA) to the A-site through elongation factor Tu (EF-Tu) (eEF1A in eukaryotes) in complex with GTP [START_REF] Schmeing | What recent ribosome structures have revealed about the mechanism of translation[END_REF][START_REF] Voorhees | Structural basis of the translational elongation cycle[END_REF]. Only after the accommodation of the correct aa-tRNA, peptide bond formation takes place. Peptide-bond formation occurs by transferring the peptidyl chain from the P-site tRNA to the aa-tRNA A-site (Blanchard et al. 2004a;[START_REF] Sanbonmatsu | Simulating movement of tRNA into the ribosome during decoding[END_REF]. This is followed by the translocation process that involves the displacement of the incoming tRNA and moving the tRNAs from the A-and P-sites to the Pand E-sites. Translocation is catalyzed by EF-G (eEF2 in eukaryotes) in complex with GTP (Yamamoto et al. 2014, Fig. 10).

Figure 10. Conservative elongation process of protein synthesis in all kingdoms. Here is an example of this process for prokaryotes.

Termination and recycling

The elongation phase continues until a stop codon is. The recognition of the stop codon is assured by ribosomal release factors (RF). Release factors 1 and 2 hydrolyze the peptidyl-tRNA bond and thereby dissociate the polypeptide chain and the ribosome [START_REF] Klaholz | Molecular recognition and catalysis in translation termination complexes[END_REF][START_REF] Rodnina | The ribosome as a versatile catalyst: Reactions at the peptidyl transferase center[END_REF]). In turn, RF3 catalyzes the dissociation of RF1 and RF2 in the GTP-dependent manner. During the post-termination process, the complex is disassembled thanks to EF-G, ribosomal recycling factor (RRF) and IF3 [START_REF] Freistroffer | Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner[END_REF][START_REF] Koutmou | RF3:GTP promotes rapid dissociation of the class 1 termination factor[END_REF]Peske et al. 2014, Fig. 11). After the recycling phase a new round of translation initiation may begin. In eukaryotes, termination and recycling processes are different. The termination process begins with the appearance of a stop codon (UGA, UGG and UAG) in the mRNA sequence. The eukaryotic termination factor 1 (eRF1) mimics tRNA and with eukaryotic termination factor 3 occupy the A-site of the ribosome. ABCE1 triggers ribosome disassembly into subunits by an ATP hydrolysis (Becker et al., 2012, Fig. 12). As the project of the current thesis is focused on the initiation process, it will be detailed more exhaustively than the other phases of mRNA translation regulation. The process of translation initiation is a tightly controlled phase and is the target of many regulatory mechanisms important for cellular physiology, viral infections and other diseases. This process has been studied for many years by different groups, but yet still not fully understood.

Two renowned research groups, Marina Rodnina and Venki Ramakrishnan, have set the pace in this domain and published highly significant results that I will briefly discuss. The group of Marina Rodnina studied the protein binding pathway using Förster resonance energy transfer (FRET) experiments. The results of their FRET studies have concluded a certain assembly sequence according to which it is the mRNA that binds first to the small ribosomal subunit, followed indifferently by IF2 and IF3, and only after IF1 then fMettRNA fMet can join (Milon et al., 2012, Fig. 15). (Milon et al., 2012).

Four years later, an article from the Venki Ramakrishnan's group presented significantly different conclusions from those described by the Rodnina group. Indeed, they find that the process commences similarly by the binding of the mRNA, after which IF1 and IF3 bind to the 30S-mRNA complex, followed by the simultaneous binding of IF2 and fMettRNA fMet to the initiation complex (Hussain et al., 2016, Fig. 16).

Such significant discrepancies highlights a lack of clear understanding of how the formation of the 30S IC occurs.

Translation initiation in bacteria

Figure 16. The assembly of the initiation complex, based on cryo-EM structures [START_REF] Hussain | Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation[END_REF].

Components of initiation complex in bacteria IF1

Initiation factor 1 (IF1) is an 8 kDa protein that binds in the 30S A-site and is encoded by infA gene (Fig. 17). Its homologs are aIF1A in archaea and eIF1A in eukaryotes [START_REF] Kyrpides | Universally conserved translation initiation factors[END_REF][START_REF] Sørensen | Remarkable conservation of translation initiation factors: IF1/eIF1A and IF2/eIF5B are universally distributed phylogenetic markers[END_REF][START_REF] Gaur | A single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors[END_REF]. The structure of IF1 from E. coli was determined by NMR in 1997 [START_REF] Sette | The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif[END_REF]. Four years later IF1 in complex with small ribosomal subunit was crystallized [START_REF] Carter | Crystal structure of an initiation factor bound to the 30S ribosomal subunit[END_REF].

IF1 functions

Several functions of IF1 have been shown during protein synthesis. IF1 appears to interact synergically with IF2, as both of these factors stimulate reciprocally their binding to the ribosome [START_REF] Stringer | Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli[END_REF][START_REF] Weiel | The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization[END_REF][START_REF] Celano | Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes[END_REF][START_REF] Gualerzi | Initiation of mRNA translation in prokaryotes[END_REF]. IF1, together with IF2, is selective in recognizing fMet-tRNAfMet from deacylated and deformylated tRNA (Antoun et al., 2006a). Moreover, these proteins play a role in increasing the binding of mRNA to the initiation complex [START_REF] Studer | Unfolding of mRNA secondary structure by the bacterial translation initiation complex[END_REF]. IF1 was characterized from three bacterial species, E. coli, T. thermophilus and Bacillus stearothermophilus [START_REF] Hershey | Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli[END_REF][START_REF] Wolfrum | Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3[END_REF][START_REF] Kapralou | Characterization of Bacillus stearothermophilus infA and of its product IF1[END_REF].

Interestingly, as thermophilic IF2 compare to E. coli has a shorter N-terminal domain, IF1 does not promote binding of IF2 to the 30S ribosomal subunit [START_REF] Kapralou | Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1-IF2 interaction[END_REF].

IF2

Initiation factor 2 (IF2) is the largest bacterial initiation factor and it includes several domains. In E. coli, IF2 is 890 amino acids long and has a molecular weight of 97,35 kDa.

However, in S. aureus IF2 is shorter 185 amino acids and is 77 kDa. IF2 is encoded by infB and was shown to be essential [START_REF] Plumbridge | Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli[END_REF][START_REF] Laalami | A severely truncated form of translational initiation factor 2 supports growth of Escherichia coli[END_REF]. Noteworthy, IF2 and the eukaryotic factor eIF5B are homologs that possess similar functions during the initiation step. [START_REF] Choi | Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast[END_REF][START_REF] Pestova | The joining of ribosomal subunits in eukaryotes requires eIF5B[END_REF] IF2 consists of 2 large domains divided into 6 subdomain: N-terminal domain (NTD)

subdivided into N1 and N2 and the C-terminal domain (CTD) subdivided into G, III, C1 and C2. The NTD of IF2 interacts with IF1 and S12. [START_REF] Boileau | Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane[END_REF][START_REF] Julián | The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli[END_REF]. It enhances the interaction of the factor with the 30S subunit [START_REF] Caserta | Translation initiation factor IF2 interacts with the 30 S ribosomal subunit via two separate binding sites[END_REF]. In some species like T. thermophilus, N2 domain is inexistent [START_REF] Simonetti | Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor[END_REF]. This factor has a GTP binding subdomain that is highly conserved among all species [START_REF] Steffensen | E. coli translation initiation factor IF2--an extremely conserved protein. Comparative sequence analysis of the infB gene in clinical isolates of E. coli[END_REF][START_REF] Simonetti | Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor[END_REF]. The CTD recognizes and binds the fMet-tRNA fMet through C2. No particular function for C1 was shown yet [START_REF] Guenneugues | Mapping the fMet-tRNA(f)(Met) binding site of initiation factor IF2[END_REF], Spurio et al., 2000, Szkaradkiewicz et al., 2000, Fig. 18

)
The GTPase activity

The GTPase activity of IF2 depends on the presence of an attached ribosome [START_REF] Luchin | In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling[END_REF]. Guanosine triphosphate (GTP) hydrolysis is triggered by 50S subunit joining to the 30S initiation complex [START_REF] Luchin | In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling[END_REF][START_REF] Tomsic | Late events of translation initiation in bacteria: a kinetic analysis[END_REF].

IF2 functions

IF2 is a protein that is associated with the ribosome throughout the entire initiation process, participating initially in the formation of the 30S initiation complex and subsequently in the assembly of the 70S initiation complex [START_REF] Milon | The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor[END_REF]. It is responsible for recognition of the formyl group of fMet-tRNA fMet and in combination with GTP is able to recruit fMet-tRNA fMet to the small ribosomal subunit, ensuring the proper location of the initiator tRNA in the ribosomal P-site [START_REF] Lockwood | A complex between initiation factor IF2, guanosine triphosphate, and fMet-tRNA: an intermediate in initiation complex formation[END_REF][START_REF] Majumdar | Specific binding of Excherichia coli chain Initiation factor 2 to fMet-tRnafMet[END_REF][START_REF] Sundari | Interaction of bacterial initiation factor 2 with initiator tRNA[END_REF][START_REF] Teana | Late events in translation initiation. Adjustment of fMet-tRNA in the ribosomal P-site[END_REF]. It was shown that IF2 participates not only in translation initiation process. It also assists in protein folding and renaturation in the cytoplasm [START_REF] Caldas | Chaperone properties of bacterial elongation factor EF-G and initiation factor IF2[END_REF].

IF3

Initiation factor 3 (IF3) is a 20,4 kDa protein encoded by infC and has just one functional homolog, eIF1 [START_REF] Olsson | Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli[END_REF][START_REF] Sabol | Purification and properties of initiation factor F3[END_REF][START_REF] Lomakin | The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH[END_REF].

The first structure of IF3 was solved by NMR in 1994 [START_REF] Fortier | The N-terminal half of initiation factor IF3 is folded as a stable independent domain[END_REF]. A year later the structure of this protein was elucidated by X-ray crystallography [START_REF] Biou | X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix[END_REF]. It was shown that IF3 consists of 2 domains (N-and C-domains) connected by a flexible linker [START_REF] Moreau | Heteronuclear NMR studies of E. coli translation initiation factor IF3. Evidence that the inter-domain region is disordered in solution[END_REF]. Most of the know functions of IF3 are performed by the C-domain, while the N-terminal part modulates the thermodynamic stability of the 30S-IF3 complex (Garcia et al., 1995[START_REF] Petrelli | Translation initiation factor IF3: two domains, five functions, one mechanism?[END_REF] Moreover, the N-domain is involved in start codon recognition, initiator tRNA selection and inhibition of leaderless mRNA translation [START_REF] Maar | A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels[END_REF]. The C-terminal domain (CTD) binds reversibly near the P-site, while the Nterminal domain (NTD) binds first to the platform and then to the elbow of fMet-tRNA fMet [START_REF] Tapprich | Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis[END_REF], McCutcheon et al., 1999[START_REF] Dallas | Interaction of translation initiation factor 3 with the 30S ribosomal subunit[END_REF], Fabbretti et al., 2007, Julián et al., 2011, Fig 19).

IF3 functions

Initiation factor 3 prevents ribosomal subunits association by binding to the 30S subunit and does not dissociate from it before the arrival of the 50S subunit [START_REF] Antoun | How initiation factors tune the rate of initiation of protein synthesis in bacteria[END_REF][START_REF] Debey | Ribosomal subunit interaction as studied by light scattering. Evidence of different classes of ribosome preparations[END_REF], Grunberg-Manago et al., 1975). It was shown that 50S also has a noncanonical binding site for IF3 [START_REF] Goyal | Non-canonical binding site for bacterial initiation factor 3 on the large ribosomal subunit[END_REF]. It catalyzes the formation of 30S initiation complexes (30S IC) [START_REF] Wintermeyer | Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study[END_REF]. IF3 selects the initiator tRNA against elongator tRNAs in 30S IC [START_REF] Hartz | Selection of the initiator tRNA by Escherichia coli initiation factors[END_REF]. It stimulates dissociation of deacylated tRNA or incorrectly bound tRNAs from the ribosomal P-site [START_REF] Karimi | Novel roles for classical factors at the interface between translation termination and initiation[END_REF][START_REF] Peske | Sequence of steps in ribosome recycling as defined by kinetic analysis[END_REF][START_REF] Antoun | How initiation factors tune the rate of initiation of protein synthesis in bacteria[END_REF]. It is also known that IF3 discriminates against initiation of leaderless mRNAs on the 30S subunit and non-initiator codon in the P-site [START_REF] Sussman | Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo[END_REF][START_REF] Haggerty | IF3-mediated suppression of a GUA initiation codon mutation in the recJ gene of Escherichia coli[END_REF][START_REF] Meinnel | Discrimination by Escherichia coli initiation factor IF3 against initiation on non-canonical codons relies on complementarity rules[END_REF][START_REF] O'donnell | Leaderless mRNAs bind 70S ribosomes more strongly than 30S ribosomal subunits in Escherichia coli[END_REF]. In addition, IF3 stimulates the dissociation of deacylated tRNA from post-termination complexes [START_REF] Hirokawa | Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic[END_REF][START_REF] Karimi | Novel roles for classical factors at the interface between translation termination and initiation[END_REF][START_REF] Peske | Sequence of steps in ribosome recycling as defined by kinetic analysis[END_REF] and plays a role in recycling stalled ribosomal complexes [START_REF] Singh | Evidence for a role of initiation factor 3 in recycling of ribosomal complexes stalled on mRNAs in Escherichia coli[END_REF][START_REF] Singh | Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli[END_REF]. Finally, together with IF1 is involved in the reaction of the cell to cold shock conditions [START_REF] Giuliodori | Preferential translation of cold-shock mRNAs during cold adaptation[END_REF][START_REF] Giuliodori | Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias[END_REF].

fMet-tRNA fMet

initiator tRNA

There are 3 forms of tRNA in the cell: aminoacyl-tRNA, peptidyl-tRNA and deacylated tRNA, which differ depending on the acylation stage, where the amino acid or peptide is attached to the 3'-end of the tRNA or this group is missing. In bacteria, translation initiation involves a specific tRNA, the initiator tRNA (itRNA) that is capable of recognizing of alternative initiation codons (AUG, GUG, UUG) [START_REF] Rajbhandary | Initiator transfer RNAs[END_REF][START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF][START_REF] Kozak | Initiation of translation in prokaryotes and eukaryotes[END_REF][START_REF] Mayer | Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation[END_REF].

Initiator tRNA has a number of structural features:

1) The discrepancy between nucleotides 1 and 72 (C1 and A72 in E. coli) at the end of the acceptor stem which prevent binding of fMet-tRNA fMet to EF-Tu [START_REF] Rajbhandary | Initiator transfer RNAs[END_REF].

2) Purine:pyrimidine base pairs 11:24 are replaced by pyrimidine:purine in elongation tRNA [START_REF] Varshney | From elongator tRNA to initiator tRNA[END_REF].

3) The presence of three conserved G:C base pairs at the bottom of the anticodon stem that make the anticodon stem less flexible compared to the anticodon stem loops of elongator tRNAs which favors biding of the tRNA fMet to the P site on the ribosome [START_REF] Cory | Primary structure of a methionine transfer RNA from Escherichia coli[END_REF], Dube et al., 1968[START_REF] Rich | Transfer RNA: molecular structure, sequence, and properties[END_REF][START_REF] Seong | Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop[END_REF][START_REF] Schweisguth | On the conformation of the anticodon loops of initiator and elongator methionine tRNAs[END_REF].

Aminoacylation

Aminoacylation of tRNA is carried out by aminoacyl-tRNA synthetases. In the case of initiator tRNA fMet , this function is performed by methionyl-tRNA synthetase (MetRS). The anticodon plays a crucial role in the recognition of tRNA fMet by MetRS (Schulman 1991).

Aminoacylation will not take place if the anticodon of initiator tRNA is mutated [START_REF] Fourmy | Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase[END_REF][START_REF] Mechulam | Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features[END_REF].

Formylation

Formylation of Met-tRNA fMet in bacteria, chloroplasts and mitochondria is catalyzed by methionyl-tRNA formyltransferase (MTF) with the participation of the N10formyltetrahydrofolate (formyl-N10-THF) as a donor of the formyl group (Schmitt et al., 1996a(Schmitt et al., , 1996b[START_REF] Schmitt | Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet[END_REF]. The marker by which MTF recognizes initiator tRNA is the non-Watson-Crick base pair 1:72 in tRNA fMet . [START_REF] Schmitt | Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet[END_REF] Initiator tRNA also differs from elongator by the presence of the formyl group that prevents binding to EF-Tu and facilitates the selection of fMet-tRNA fMet by IF2 [START_REF] Sundari | Interaction of bacterial initiation factor 2 with initiator tRNA[END_REF][START_REF] Hansen | Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli[END_REF][START_REF] Rajbhandary | Initiator transfer RNAs[END_REF][START_REF] Nissen | Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog[END_REF][START_REF] Kozak | Initiation of translation in prokaryotes and eukaryotes[END_REF].

mRNA

Messenger RNA (mRNA) is an RNA containing information about the amino acid sequence of proteins, which is synthesized on the basis of DNA during transcription process, after which is used during translation stage as a matrix for protein synthesis.

Single-stranded RNAs can bind to 30S with high affinity even in the absence of initiation factors and fMet-tRNA fMet (Kozak, 1999, De Smit and[START_REF] De Smit | Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA[END_REF]. In E. coli, it has been shown that ribosomal protein S1 is involved in this process. It unfolds highly structured mRNAs, making binding between 30S and mRNA more stable [START_REF] Duval | Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation[END_REF]. Also, mRNA has A/U-rich elements which are recognized by protein S1 [START_REF] Steitz | Polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage R17 RNA[END_REF][START_REF] Dreyfus | What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs?[END_REF][START_REF] Jin | Influences on gene expression in vivo by a Shine-Dalgarno sequence[END_REF].

Translation initiation region

The mRNA contains a combination of several elements located in translation initiation region (TIR) affecting the efficiency of initiation process that are the start codon, the Shine-Dalgarno sequence and the spacer between the SD sequence and the start codon. The role of TIR is to proper position the mRNA on the 30S subunit for translation initiation [START_REF] Dreyfus | What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs?[END_REF].

The Shine-Dalgarno sequence (SD)

The Shine-Dalgarno sequence is a purine-rich sequence consisting of 4-8 nucleotides and separated from the start codon by a 5-13 spacer [START_REF] Schneider | Information content of binding sites on nucleotide sequences[END_REF]. The SD complementary to the anti-Shine-Dalgarno sequence (aSD) located at the 3'-end of 16S rRNA [START_REF] Shine | The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites[END_REF]. In E. coli, a typical SD sequence is GGAGG and most common spacer length is 7-10 nucleotides long [START_REF] Ma | Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures[END_REF].

The start codon

There are several types of start codons. In E. coli, 83% of the total is AUG codon [START_REF] Ma | Correlations between Shine-Dalgarno sequences and gene features such as predicted expression levels and operon structures[END_REF]. The remaining 14% is GUG and 3% is UUG codons. Rare non-canonical codons AUU, AUC and CUG have been reported [START_REF] Schneider | Information content of binding sites on nucleotide sequences[END_REF][START_REF] Polard | Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911[END_REF][START_REF] Mccarthy | Prokaryotic translation: the interactive pathway leading to initiation[END_REF][START_REF] Binns | Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU[END_REF][START_REF] Baudet | Proteomics-based refinement of Deinococcus deserti genome annotation reveals an unwonted use of noncanonical translation initiation codons[END_REF].

The primary project of the current thesis is to study mRNA translation initiation in S.

aureus. Staphylococci are gram-positive cocci (~1 μm in diameter) that cause many forms of infections. They grow and together they form clusters, pairs and occasionally short chains.

The configuration of the cocci helps to distinguish micrococci and staphylococci from streptococci, which usually form chains. One of the most significant representatives is

Staphylococcus aureus that tends to localize in the nose, respiratory tract and on the skin upon transmission. One fifth of the human populations in the world suffer from diseases caused by S. aureus. These organisms can grow aerobically or anaerobically at temperatures between 18 °C and 40 °C and in a pH range from 4.2 to 9.3 [START_REF] Le Loir | Staphylococcus aureus and food poisoning[END_REF][START_REF] Taylor | Staphylococcus Aureus. StatPearls[END_REF].

Pathogenic strains produce virulence factors, such as potent protein toxins that damage biological membranes, leading to cell death. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. One of the most representative examples are enterotoxins causing food poisoning and when expressed systemically, can cause toxic shock syndrome [START_REF] Lyon | Antimicrobial resistance of Staphylococcus aureus: genetic basis[END_REF]Skurray 1987, Prevost et al., 1995).

In staphylococci, the ability to respond to cell density with genetic adaptations is due to one main system called "accessory gene regulator" (Agr) [START_REF] Le | Quorum-sensing regulation in staphylococci-an overview[END_REF]. It is a key to the temporal regulation of adhesins and exotoxins and is consists of two divergent transcripts, RNAII encodes a quorum sensing cassette (AgrBD) and a two-component system (AgrAC)

while RNAIII is the multifunctional RNA, which encodes a PSM δ-hemolysin (hld). [START_REF] Novick | Quorum Sensing in Staphylococci[END_REF]. RNAIII is a 514-nt highly abundant and stable regulatory RNA. Its structure, but not its sequence, is well conserved among several staphylococcal species [START_REF] Benito | Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression[END_REF].

The main function of RNAIII is regulating translation of both individual exoproteins and pleiotropic regulators [START_REF] Novick | Quorum Sensing in Staphylococci[END_REF]. For example, RNAIII represses spa gene expression that codes for the surface protein A, one of the major virulence factors, interfering with Igmediated opsonization [START_REF] Patel | Virulence of protein Adeficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement[END_REF] and promoting collagen mediated adhesion to connective tissues by its interaction to the von Willebrand factor [START_REF] Hartleib | Protein A is the von Willebrand factor binding protein on Staphylococcus aureus[END_REF], Gomez at al., 2004). The 3' end of RNAIII is partially complementary to the 5' end of spa mRNA.

Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. (Huntzinger, 2005, Fig. 21)

Translation regulation in S. aureus

Figure 21. RNA-mediated mechanisms used in virulence gene regulation. The mechanism of action of S. aureus RNAIII on spa expression [START_REF] Romby | The role of RNAs in the regulation of virulence-gene expression[END_REF].

Specific features of the protein synthesizing apparatus of Staphylococcus aureus

Despite the fact that ribosomes of different bacterial species are very similar, there are many species-specific peculiarities that can reflect on various vital processes, including the process of translation initiation. These unique species-specific differences are associated with structural variations, specific for each species. For example, uS9, uL3 and bL31 in S. aureus, uS14 in E. coli and uS8, bL19 and bL28 in T. thermophilus present extensions that protrude from the ribosome. Similarly, for the rRNA, the presence of short insertions and deletions in the 16S and 23S rRNA are also common species-specific features. For instance, helix 6, helix 10, helix 26 (h26) and helix 44 in the small subunit of the ribosome, and also Helix 28 and Helix 68 in the large subunit have different lengths or adopt different folds and orientations.

h26 on the SSU participates in the ribosome's dimerization during the hibernation process in S. aureus by creating contacts between two SSU belonging to two different ribosomes. This helix is longer than in S. aureus when compared to other known bacterial species. In gramnegative bacteria like T. thermophilus this helix is shorter by 3 base pairs, making contact between the ribosomes impossible without additional rotation [START_REF] Khusainov | Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF[END_REF].

Another striking example of ribosome variability is protein bL31 that bridges two subunits where it interacts with uS13, uS19, uL5 and 5S rRNA. There are 2 types of this protein: type A and B. In S. aureus, only type B is present (type A is not encoded), which contains an additional loop, instead of a zinc-binding motif. For the rest type B is absolutely analogous to type A, namely it includes a three-stranded beta sheet near the N-terminus and a C-terminal extension, which may contain an alpha helix. In general, bL31 can adapt to the large-scale movements of the ribosome important for tRNA proofreading [START_REF] Jenner | Structural rearrangements of the ribosome at the tRNA proofreading step[END_REF][START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF].

S1

S1 is an essential and atypical ribosomal protein that plays a major role in unfolding highly structured mRNAs before binding to the ribosome. Depends on the organism S1 may have different functions and play a different role during the translation initiation process. In E. coli it consists from 6 domains: 1st and 2nd are involved in ribosome binding, the rest 4

have been shown to interact with mRNA [START_REF] Subramanian | Graphite furnace atomic absorption spectrometry with matrix modification for determination of cadmium and lead in human urine[END_REF].

Several studies have been conducted related to the replacement of S1 protein for the same protein from another organism. It was shown that the addition of S1 from E. coli or from

Micrococcus luteus to depleted S1 ribosomes of E. coli restored 30S properties for translation of mRNA carrying weak SD, but these proteins did not affect the B. subtilis ribosomes. Since these proteins are similar in B. subtilis and S. aureus and have lost the N-terminal domain, which has been shown to promote specific binding to S2 in E. coli 30S, it may explain why this protein does not play a major role in the translation process (Farwell et al., 1992, Vellanoweth andRabinowitz, 1992).

It was also shown that B. subtilis ribosomes have a higher tolerance to non-AUG initiation codons than E. coli ribosomes. The presence of mRNA with a strong SD sequence also significantly increases the ability to bind to the ribosome. In this regard, it was suggested that firmicutes eliminates the need for S1 protein acting on 30S due to the presence of a large amount of mRNA with a strong SD [START_REF] Omotajo | Distribution and diversity of ribosome binding sites in prokaryotic genomes[END_REF].

Initiation factors

The amino acid sequences of initiation factors 1 and 3 from three bacterial species Antibiotics may act on one or more different steps in bacterial metabolism. Most of known antibiotics act on cell wall synthesis (penicillins and cephalosporins), while others act on protein synthesis [START_REF] Ghooi | Inhibition of cell wall synthesis--is this the mechanism of action of penicillins?[END_REF]. In turn, there are a number of mechanisms allowing bacteria to resist the action of antibiotics.

Selective membrane permeability

The major antibiotics penicillins, cephalosporins, monobactams and glycopeptides (vancomycin and teicoplanin), also called β-lactams inhibit bacterial cell wall synthesis by preventing synthesis of peptidoglycan, which is an important constituent of the bacterial cell wall [START_REF] Ghooi | Inhibition of cell wall synthesis--is this the mechanism of action of penicillins?[END_REF]Thatte, 1995, Trevor et al., 2015). This group of antibiotics combines the presence of a β-lactam ring that can be hydrolyzed by the action of enzymes (β-lactamase), making these drugs ineffective [START_REF] Bondi | Penicillin resistant staphylococci[END_REF]. Given the high clinical efficacy and low toxicity, β-lactam antibiotics form the basis of antimicrobial chemotherapy at the present stage, taking the leading place in treating most infections.

Protein synthesis inhibitors

In recent years, interest in the ribosome as a target for the discovery of new antibacterial agents has increased and was caused by defining the structure of whole bacterial ribosomes, subunits, domains and antibiotic complexes (Hermann Review, 2005, Tenson and[START_REF] Tenson | Antibiotics and the ribosome[END_REF]. The advent of three-dimensional structures confirmed early biochemical studies that antibiotics interact with the bacterial ribosome RNA. Resistance to this family of antibiotics are partly due to changes in the drug-binding sites (chemical modification and/or change in conformation). Mutations and/or deletions of ribosomal proteins can also cause resistance, but since ribosomal proteins usually do not interact directly with the drug, it is believed that resistance occurs indirectly, causing conformational changes in rRNA (Schlunzen et al., 2001[START_REF] Tu | Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance[END_REF][START_REF] Davidovich | Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity[END_REF].

Antibiotics

Antibiotics targeting the translation apparatus may act on certain functional steps -some block the termination, others initiation but most of them inhibit elongation [START_REF] Wilson | The A-Z of bacterial translation inhibitors[END_REF][START_REF] Sohmen | SnapShot: Antibiotic inhibition of protein synthesis I[END_REF].

For instance, the formation of 70S can be inhibited by pactamycin on the 30S subunit or by avilamycin on the 50S subunit. compounds specifically targeting the initiation phase are limited in terms of clinical application owing to problems with drug specificity (edeine), toxicity (evernimicin) and solubility (thiopeptides) [START_REF] Dinos | Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site RNA binding[END_REF][START_REF] Belova | A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit[END_REF][START_REF] Mikolajka | Differential effects of thiopeptide and orthosomycin antibiotics on translational GTPases[END_REF]. As for the inhibition of elongation, streptomycin and tetracyclines prevent the delivery of aminoacylated-tRNA (aa-tRNA) to the A-site of the ribosome (Day, 1966[START_REF] Cox | Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid[END_REF]. Chloramphenicol, linezolid and puromycin interfere with the peptidebond formation between the A-and P-site tRNAs [START_REF] Cundliffe | Bacterial protein synthesis: the effects of antibiotics[END_REF][START_REF] Monro | Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide[END_REF], Kalpaxis at el., 1989[START_REF] Skripkin | R chi-01, a new family of oxazolidinones that overcome ribosome-based linezolid resistance[END_REF]. Translocation of the tRNAs is inhibited by aminoglycoside and elongation of the nascent chain is inhibited by the macrolides (Walsh, 2003). While all of the above-mentioned antibiotics bind on the 30S, some elongation inhibitors also target the 50S. Finally, the peptidyl-transferase inhibitors (chloramphenicol) prevent termination, whereas recycling is inhibited by translocation inhibitors (fusidic acid) [START_REF] Caskey | Hydrolysis of fMet-tRNA by Peptidyl Transferase[END_REF], Savelsbergh et al., 2009, Fig 23, Table 3).

Antibiotics that inhibit the synthesis of nucleic acids

Quinolones are synthetic antibiotics that target DNA gyrase as their site of action [START_REF] Lesher | 1,8-Naphthyridine derivatives. A new class of chemotherapeutic agents[END_REF]. There are three mechanisms of action for these antibiotics: (1) by developing point mutations in the targets, (2) by decreasing the intracellular concentration of the quinolone and (3) by acquisition of mobile elements carrying the qnr gene, which confers resistance to quinolones [START_REF] Ruiz | Transferable mechanisms of quinolone resistance[END_REF].

Antibiotics that inhibit RNA synthesis include rifamycin, which binds to RNA polymerase, an enzyme that catalyzes the process of transcription. It is a broad-spectrum antibiotic that inhibits both gram-negative and gram-positive. Rifamycin is often used in combination with other antibiotics, since bacteria quickly develop resistance to it by acquiring point mutations in RNA polymerase that prevent the binding of the antibiotic [START_REF] Hartmann | The specific inhibition of the DNA-directed RNA synthesis by rifamycin[END_REF][START_REF] Campbell | Structural mechanism for rifampicin inhibition of bacterial rna polymerase[END_REF].

Fatty acid synthesis inhibitors

Fab-001 (MUT056399; FAB Pharmaceuticals) and AFN-1252 (Affinium Pharmaceuticals) are molecules that are in clinical trials. It has already been shown that these molecules are highly effective against several strains of MRSA [START_REF] Payne | Discovery of a novel and potent class of FabI-directed antibacterial agents[END_REF][START_REF] Escaich | The MUT056399 inhibitor of FabI is a new antistaphylococcal compound[END_REF].

Current drugs against S. aureus

Staphylococcus aureus is an extremely adaptive pathogen with a proven ability to develop resistance. Despite the presence of a large number of antibiotics, there is a need to develop new compounds more effective and less toxic to fight against this organism [START_REF] Chambers | Waves of resistance: Staphylococcus aureus in the antibiotic era[END_REF]. After having introduced the above-mentioned concepts, I will present now my two projects of this thesis.

While the ribosomal structures of gram-negative bacteria like Thermus thermophilus and Escherichia coli were solved a long time ago, the structure of gram-positive bacteria was published only 3 years ago, despite the practical significance of this group, since most pathogens to humans are gram-positive bacteria.

Pathogenic bacteria like Staphylococcus aureus produce an extremely high resistance to antibiotics, which mainly affect the process of protein synthesis. It is also known that the process of protein synthesis in gram-positive and gram-negative bacteria has significant differences. Therefore, for a better understanding of the mechanism of antibiotic resistance, it is necessary to study this process in detail.

Moreover, pathogenic bacteria have complex translation regulation mechanisms that allow them to survive under stressful conditions.

Aim of the project:

The main goal of this project is to study the initiating process in S. aureus, a rate-limiting step that includes the action of many precisely regulated components.

It is necessary to obtain high-resolution structures in order to gain further insight on this finely regulated pathway and possible species-specific differences of this process.

Project 1: Assembly pathway of the S. aureus initiation complex

One of the most common human fungal pathogen is a Candida albicans, causing from superficial, cutaneous-mucosal to deep-seated and disseminated infections [START_REF] Segal | Candida, still number one--what do we know and where are we going from there?[END_REF]. It has been found in at least 70% of the human population [START_REF] Ruhnke | Management of mycoses in patients with hematologic disease and cancer --review of the literature[END_REF].

There is a definite correlation between the host and the microbes in normal conditions, with an equilibrium shift towards C. albicans leads to the emergence of various diseases and their aggravation in the presence of immunodeficiency [START_REF] Sardi | Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options[END_REF].

Treatment of diseases caused by this organism costs about 2-4 billion dollars in the United States (Perloth et al., 2007). The ineffectiveness of the treatment is due to the occurrence of serious side effects associated with the fact that the cellular targets in C. albicans are often shared with humans [START_REF] Berman | Candida albicans: a molecular revolution built on lessons from budding yeast[END_REF].

Thus, there is a need to study the physiological mechanisms of C. albicans, with the further use of the knowledge gained in the development of new drugs in the fight against this pathogen.

Candida's cells can grow in a wide range of conditions that can affect cell shape. They grow at pH from 2 to 8 and from 20 to 40 o C. The optimum temperature is 37 degrees. At low pH (less than 6), the cells grow in an ovoid-shaped budding type, but when the pH>7, hyphal growth is induced [START_REF] Odds | Candida and candidosis[END_REF]. This organism is a facultative anaerobe that at least 4 times more resistant to antifungal treatments in anaerobic than in aerobic conditions [START_REF] Dumitru | Defined anaerobic growth medium for studying Candida albicans basic biology and resistance to eight antifungal drugs[END_REF].

Specific features of Candida albicans ribosome

The molecular weight of Candida albicans ribosome is 3.2 MDaltons. This ribosome consist of 44 r-proteins in the large subunit and 35 in the small subunit. The ratio of rRNA to r-proteins is 1.27 (Annex) (Bruchlen D. thesis).

Using bioinformatics methods, it was shown that A-and P-sites between С. albicans, Saccharomyces cerevisiae (S. cerevisiae) and Homo sapiens are conserved (Bruchlen D. thesis). A significant difference was found in the E-site between С. albicans and S. cerevisiae that could explain the resistance of C. albicans to cycloheximide (CHX) [START_REF] Goldway | Multidrug resistance in Candida albicans: disruption of the BENr gene[END_REF].

Project 2: Crystallogenisis of Candida albicans 80S

CHX is a known inhibitor of the eukaryotic translation that binds on the E-site of ribosome without any conformational modifications of the residues. It inhibits the mRNA translation mechanism by stalling translating ribosomes on mRNA. It also blocks the release of tRNA after their delivery of the new amino acids (Garreau de [START_REF] Garreau De Loubresse | Structural basis for the inhibition of the eukaryotic ribosome[END_REF].

The size of the E-site binding pocket is smaller due to a modification of 56 amino acid of the eL42 r-protein. The modification is associated with the replacement of proline in S.

cerevisiae by glutamine in C. albicans, causing a steric clash that is certainly implicated in this CHX resistance (Bruchlen D. thesis).

Another feature of the species is connected with the eS26 protein, which is longer in fungi compared to protozoa and C-terminal region of this protein is different from the metazoan group (Bruchlen D. thesis).

Aim of the project:

This project was started by David BRUCHLEN in 2012 in Marat Yusupov's laboratory (IGBMC, University of Strasbourg) when the ribosome structure from pathogens was not yet determined. Due to the similarity of Candida albicans and Saccharomyces cerevisiae, it was decided to start using the purification and crystallization protocols that have already been developed in the laboratory.

Despite the similarity of these yeasts, it was not possible to obtain crystals under these conditions. David spent 4 years to obtain crystals with high-resolution diffraction. Once, he managed to get the structure of 3.7 Å, which is not enough to study the effect of antibiotics against this organism. Moreover, these crystallization conditions were not reproducible.

My goal in this project was to optimize the protocols of purification and crystallization of the ribosome C. albicans, make them reproducible and further improve the resolution of the ribosome structure needed to study the effect of antibiotics. Now this project is still ongoing by my colleagues in Marat Yusupov's laboratory.

RESEARCH PROJECT 1

Translation initiation complex from S. aureus

MATERIALS AND METHODS

70S PURIFICATION

Buffers were used during the experiment:

In vitro reconstruction of translation initiation complex

Staphylococcus aureus cell growth

For our experiments we used the RN6390 strain for fast and efficient cell lysis. In this strain the production of RNAIII is increased and the biofilms formation is decreased.

All experiments were carried out in a special laboratory with safety level 2. A brain heart infusion medium (BHI) was used for cell growth. Cells were grown at 37 o C and harvested in the early logarithmic phase (OD 600 = 1 OD/ml), when ribosomes are more active and be found in maximum concentration (Gourse et el., 1996). Cells were washed in buffer A and kept frozen at -80 o C, due to subsequent long-term experiments.

The protocol of full ribosome (70S) purification, which will be described below, was developed by Iskander Khusainov in the laboratory of Marat Yusupov (IGBMC, University of Strasbourg) [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF].

Lysis

For cell lysis, we used the antibacterial enzyme lysostaphin, which is capable of cleaving pentaglycin cross-linking bridges found in Staphylococci. We could not use bulky equipment like French press and microfluidizer due to lack of space in a special laboratory with safety level 2.

Frozen cells were resuspended in buffer A containing 1 mM EDTA, lysostaphin, DNAse I and protease inhibitor cocktail (Roche) and then incubated at 37 o C for 45 minutes in a water bath. All subsequent procedures were performed at +4 o C or on ice. Cell debris was removed by centrifugation at 30000 g for 90 minutes.

PEG precipitation

This step was taken from the yeast ribosome purification protocol and is necessary for the gentle precipitation of ribosomes from the cell extract.

PEG20K (stock 30% w/v from Hampton research) was added to the supernatant to final concentration 2.8% w/v (5 minutes, 4 o C). The resulting solution was centrifuged 20000 g for 5 minutes. The supernatant containing the ribosomes and light cellular components were collected and PEG 20K was added to it till the final concentration of 4.2% (10 minutes, 4 o C).

The resulting solution was centrifuged at 20000 g for 10 minutes. At this stage, ribosomes are precipitated, while everything that is lighter remains in the supernatant.

Sucrose cushion

This is a common step for purification of the vacant ribosome. It includes to use the sucrose cushion with 0.5-1 M salt concentration. This step allows us to remove from the sample mRNA, tRNA, translation factors and other proteins bound to the ribosome.

The ribosomal pellet obtained in the previous step was resuspended in 35 ml of buffer A containing 1 mM EDTA and layered to 25 ml cushion of buffer B. After this, the solution was centrifuged at 45000 rpm for 15 hours using the Beckman Type 45 Ti rotor.

Sucrose density gradient centrifugation

The idea of this step was to separate the full ribosomes (70S) from dimers (100S) and subunits, due to size and weight.

The ribosomal pellet from sucrose cushion was resuspended in buffer E, loaded on 7% -30% w/v sucrose gradients and centrifuged at 17100 rpm for 15.5 hours on swinging the rotor Beckman SW28, 3,5 mg of ribosomes per tube. (Fig. 24).

Last PEG precipitation

Selected fractions were pooled and the concentration of Mg(OAc)2 was adjusted to 25 mM. Further, PEG20K was added to the final concentration 4.5% w/v to precipitate the ribosome (10 minutes, 4 o C) and the solution was centrifuged at 20000 g for 12 minutes. After this, the pellet was resuspended in buffer G and used later for small ribosomal subunit purification.

The purity and quality of the obtained sample were checked by several methods: protein PAGE, agarose gel in native and denaturing conditions, mass spectrometry and crystallization.

One-dimensional polyacrylamide gel (PAGE)

One-dimensional PAGE in SDS denaturing conditions separates proteins by molecular weight. Using this method, information about the presence of non-ribosomal proteins in the sample can be obtained.

The gel demonstrates the purity of the sample and the absence of high molecular weight contaminants like pyruvate dehydrogenase complex, usually co-purified with bacterial ribosome (Fig. 25).

Agarose gel in native and denaturing conditions

The native agarose gel contains 0.8% agarose and TBE buffer (90 mM tris-HCl pH 8.3, 89 mM boric acid and 10 mM EDTA pH 8, pH was adjusted till 8.3 by HCl). Guanidine thiocyanate was added to the sample till the final concentration of 10 mM to obtain denaturing conditions.

On the agarose gel in native conditions, two subunits of 30S and 50S are shown. In denaturing conditions, the integrity of ribosomal RNA of both 16S and 23S was demonstrated (Fig. 26).

Mass spectrometry

Mass spectrometry is an analytical technique that used to identify molecules in the mixture. Biologists use this method to analyze a purified protein, detect impurities in the sample and to study the protein content of the sample of cells. In comparative proteomics studies, one or both of two parameters are generally used: a spectral count, which is the total number of spectra identified for the protein or some measure of ion abundance. LC-MS/MS analysis is a practical semi quantitative technique with which you can establish the presence or absence of proteins in extreme cases [START_REF] Milac | Analyzing LC-MS/MS data by spectral count and ion abundance: two case studies[END_REF][START_REF] Lundgren | Role of spectral counting in quantitative proteomics[END_REF].

Analysis of S. aureus ribosome sample shows the presence of all ribosomal proteins except S1 (Table 5) and a low amount of non-ribosomal proteins which indicates the purity of the sample (Table 6).

Sucrose density gradient centrifugation

After obtaining 70S, the sample in buffer G was loaded on 0-30% w/v sucrose gradients centrifuged at 21600 rpm for 14.5 hours using a Beckman SW28 rotor (Fig. 28).

Concentration and storage of the sample

Selected fractions were pooled and the concentration of Mg(OAc)2 was adjusted to 10 mM. In addition, spermidine was added to a final concentration of 2.5 mM. The sample was concentrated using centricons 100K simultaneously changing buffer G to buffer D. The sample was frozen in liquid nitrogen and stored at -80 o C.

One-dimensional PAGE in denaturing conditions

The gel shows the absence of contaminants with the high molecular weight (Fig. 29).

Agarose gel in denaturing conditions

The gel shows the absence of rRNA (16S) degradation (Fig. 30).

Toeprinting assay

Toe-printing assay is a method that allows us to examine the interactions between mRNA and ribosome. This method requires reverse transcriptase, which in the absence of a ribosome, would simply create a complete cDNA copy of mRNA. However, when the reverse The toeprint signal is increasing with the increasing ribosome concentration, which proves the ability of mRNA bind to the ribosome (Fig. 31).

INITIATION FACTORS PURIFICATION

IF1 and IF3 purifications

Buffers for IF1 purification:

Buffer A:

Cell growth

The vector pnEAvH containing infA/infC gene from S. aureus was transformed into E.

coli BL21(DE3). Cells were grown in LB medium with 100 µg/ml of ampicillin. The expression was induced during 3 hours with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). After cells were centrifuged and resuspended in 10 mM tris-HCl pH 7.5 buffer. Cells were stored at -80 o C.

Lysis

Cells were thawed for 40 minutes and then resuspended in buffer A with DNAse I and protease inhibitor cocktail (without EDTA). After, the cells were destroyed using emulsiflex and centrifuged at 20000 g for 1 hour.

Affinity chromatography

The principle of affinity chromatography is the ability of a his-tag protein to bind to nickel beads due to the affinity of polyhistidine tag to metal ions, followed by elution of the protein of interest with a competitive molecule like imidazole.

Dialysis

The decrease salt and imidazole concentrations was performed using dialysis method overnight at +4 o C. Also, during dialysis, the his-tag of IF1 was cleaved with tev protease.

For IF3, we skipped this step because of its low efficiency.

Ion exchange chromatography

To obtain the sample with high purity, an ion exchange chromatography step was added.

Since IF1 has an isoelectric point (pI) of 6.72 and IF3 9.7 a cation-exchange chromatography column monoS was used. The sample was eluted gradually by increasing the salt concentration from 100 mM till 1 M. After several purifications of IF3, cation exchange chromatography was replaced by gel filtration (Superdex 75 16/600) to get more sample.

Storage

The final sample was frozen in liquid nitrogen with 10% glycerol and stored at -80 o C.

was resuspended in 10 mM tris-HCl pH 7.5 buffer. Cells were stored at -80 o C until further use.

Lysis

Thawed cells were resuspended in buffer A that contains also lysozyme, DNAse I and protease inhibitor cocktail (without EDTA), sonicated and centrifuged at 15000 g for 30 min.

Ammonium chloride was added to the collected supernatant till 1 M to precipitate ribosomes and centrifuged at 60000 rpm for 45 minutes using a Beckman Ti 70.1 rotor.

Affinity chromatography

The protein his-tag due to the affinity binds to nickel agarose beads (Qiagen). Further, the sample was eluted with a competitive molecule imidazole (100 mM).

Dialysis

The buffer change from E to Q was performed using dialysis method overnight at +4 o C.

Tev cleavage was not performed due to ability of protein without his-tag also to bind with nickel beads.

Ion exchange chromatography

An anion exchange chromatography step was added to obtain a pure sample (pI = 5.09).

The sample was eluted gradually by increasing the salt concentration from 100 mM till 1 M.

Storage

The final sample was frozen in liquid nitrogen with 10% glycerol and stored at -80 o C.

The purity of the obtained samples was checked on polyacrylamide gels.

mRNA PURIFICATION

For our experiments, we used staphylococcus specific spa mRNA which encodes major virulence factor protein A. The mRNA length is 225 nucleotides with the UUG start codon and a strong Shine-Dalgarno sequence (Fig. 33).

Cell growth

The vector pUT7 containing spa gene from S. aureus was transformed into E. coli XL1 (from Pascale Romby's laboratory). Cells were grown in LB medium with 100 µg/ml of ampicillin.

Plasmid purification

Plasmid purification was done using NucleoBond Xtra miniprep kit (Macherey nagel).

Linearization

Linearization of plasmid was performed restriction enzyme BamHI. The resulting DNA was extracted with phenol-chloroform and precipitated in absolute ethanol. Further, the sample was loaded on agarose gel to separate linearized DNA from the plasmid, with subsequent use of NucleoSpin Gel and PCR Clean-up kit (Macherey nagel) to purify the DNA from the gel.

"In vitro" transcription

The transcription was performed using T7 MEGAscript kit (Ambion). For 1 µg of DNA used:

7,5 mM ATP solution, 7,5 mM CTP solution, 7,5 mM GTP solution, 7,5 mM UTP solution, 1 U/µl RNasin 1X reaction buffer, 7,5 U/µl T7 RNA polymerase 10x reaction buffer:

44.1 M Tris-HCl pH 7.5

1 M NaCl 1 mM EDTA pH 7.5 1 mM DTT 0,1 mM Spermidine Incubated at 37 o C for 4 hours.

After 4 hours incubation, DNase TURBO was added till final concentration 2 U/µl and incubated additional 15 minutes at 37 o C. DNase TURBO is a specifically engineered hyperactive DNase that exhibits up to 350% greater catalytic efficiency than wild type DNase I (from T7 MEGAscript kit, Ambion). Then the obtained RNA was extracted with phenol-chloroform and precipitated with isopropanol.

Midi-size gel

An additional purification step is required to obtain a high purity sample. For this, a 6% polyacrylamide gel was used which was run for 3.5 hours at 300 V. The resulting RNA was eluted in a buffer with 500 mM NH4Cl, 1 mM EDTA pH 8 and 16% phenol.

Storage

The sample was stored at -20 o C until further use.

Purified mRNA was checked on a 6% polyacrylamide gel (Fig. 34).

fMet-tRNA fMet PREPARATION

Methionyl-tRNA fmet synthetase (MetRS)

MetRS is a 74 kDa synthetase that catalyzes the formation of methionyl-tRNA fMet .

Buffers were used during the experiment:

Cell growth

The plasmid pET15b overexpressing MetRS from E. coli was provided by Cheemeng Tan (Addgene plasmid #111464). Cells were grown in terrific broth medium with 100 µg/ml of ampicillin and 0,4 mM IPTG grown overnight. Then cells were centrifuged and resuspended in 10 mM tris-HCl pH 7,5 buffer.

Lysis

Thawed cells were resuspended in buffer A with DNase I and protease inhibitor cocktail and lysed using the emulsiflex. Then the sample was centrifuged at 20000 g for 1 h to precipitate cell debris.

Affinity chromatography

A sufficiently pure sample was obtained using affinity chromatography. After the his-tag protein bound to nickel beads, it was eluted with 300 mM Imidazole.

Methionyl-tRNAfMet formyltransferase (MTF)

MTF is a 30 kDa enzyme that catalyzes the attachment of the formyl group to methionyl-tRNA fMet .

Buffers were used during the experiment are the same as for MetRS.

Cell growth

The plasmid pSC101 overexpressing MTF from E. coli was provided by Cheemeng Tan (Addgene plasmid #111492). Cells were grown in LB medium with 50 µg/ml of kanamycin and 1 mM IPTG grown for 3 hours. Then cells were centrifuged and resuspended in 10 mM tris-HCl pH 7,5 buffer.

The lysis, affinity chromatography, dialysis and storage stages are the same as described previously for MetRS.

Purified MTF was checked on 15% PAGE (Fig. 36).

tRNA fMet

The protocols that will be described below have been adapted from protocols that were published in "Methods in Enzymology" [START_REF] Mechulam | Protectionbased assays to measure aminoacyl-tRNA binding to translation initiation factors[END_REF].

Buffers were used during the experiment:

Cell growth

The plasmid pBS used for overexpression of itRNA from E. coli was provided from A. Innis's laboratory already transformed into HB101 strain. Cells were grown in LB medium overnight and after centrifugation were resuspended in buffer A.

Phenol-chloroform extraction, DNA and tRNA precipitation

After thawing cells, nucleic acids were extracted with phenol-chloroform. Sodium acetate and isopropanol were added to the obtained sample to precipitate DNA. After that, tRNA was precipitated by adding more isopropanol.

Deacetylation of tRNA

The pellet was resuspended in 200 mM tris-Ac pH 9 buffer and incubated for 1 hour at 37 o C.

Then it was precipitated in absolute ethanol and resuspended in 10 mM ammonium acetate pH 5. The quality of purification of initiator tRNA was checked on a 6% polyacrylamide gel (Fig. 37).

Ion exchange and hydrophobic interaction chromatography

The first purification step was performed using anion chromatography (Q sepharose column).

Then the sample was precipitated with isopropanol and was applied to phenyl column. Finally, the sample was concentrated using centricon tubes 10K and was kept at -20 o C.

Preparation of formyl donor

10-Formyltetrahydrofolate (f-THF) is a donor of formyl group for formyltransferase reactions usually derived from folinic acid (Sigma-Aldrich). Folinic acid were dissolved in 50 mM βmercaptoethanol and 1 N HCl and incubated at room temperature for 4 hours. The amount of formyl donor was measured at 350 nm every hour. After 4 hours the sample was diluted 6 times with water and pH was adjusted to 7.5 with 2 M tris-base pH 11. The sample was kept at -20 o C.

Aminoacylation and formylation of tRNA fMet

The reaction was performed 2 times: first with S100 [START_REF] Rajbhandary | Studies on polynucleotides. XCI. Yeast methionine transfer ribonucleic acid: purification, properties, and terminal nucleotide sequences[END_REF] supernatant after centrifugation at 100000 g) and then with enzymes.

1

CRYO-ELECTRON MICROSCOPY

The main method used during this thesis project is cryo-electron microscopy (cryo-EM). In the following I will introduce briefly this technique. Cryo-EM is a powerful structural biology technique used to solve the structures of macromolecular complexes very often at atomic resolutions.

Jacques Dubochet, Joachim Frank and Richard Henderson set the bases of the technique and subsequently received the Nobel Prize in Chemistry in in 2017. Today, cryo-EM can handle even small molecules (~30 kDa [START_REF] Liu | A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold[END_REF]). Its major advantages are the ability of analyzing heterogeneous complexes with a large number of conformations [START_REF] Serna | Hands on Methods for High Resolution Cryo-Electron Microscopy Structures of Heterogeneous Macromolecular Complexes[END_REF] without the need of forming crystals, which can be extremely challenging for large and/or hydrophobic molecular assemblies.

The remarkable progress in cryo-EM

Initial results in this area were achieved with highly symmetric viral particles and large stable multicomponent complexes, such as ribosomes. At the same time, barriers related to resolution limits, the size of macromolecular targets, the lack of internal symmetry and the solubility of the sample began to appear which were subsequently overcome in many cases. Some important improvements were achieved using higher accelerating voltages (200 -300 kV) and an electron source with a field emission gun, others using a stable lens system [START_REF] Glaeser | Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects[END_REF], an improved vacuum system, and stable cryo-stages that minimize sample drift. But the major improvement was the advent of direct electron detector device (DDD) cameras which a superior detective quantum efficiency, a measure of the combined effects of the signal and noise performance of an imaging system. Along with improving the hardware of the technique (DDD, transmission electron microscopes, vitrification devices) the softwares have also made a revolutionary leap ahead. Before image processing involved many independent and time-consuming steps while now all processes are quite automated and take a small amount of time [START_REF] Mcmullan | Detective quantum efficiency of electron area detectors in electron microscopy[END_REF][START_REF] Faruqi | Electronic detectors for electron microscopy[END_REF][START_REF] Li | Electron counting and beam-induced motion correction enable nearatomic-resolution single-particle cryo-EM[END_REF][START_REF] Milazzo | Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy[END_REF].

The transmission electron microscope

The structure and principle of a transmission electron microscopy (TEM) is similar to a light microscope, but electromagnetic lenses instead of glass lenses and electrons instead of light are used.

In the TEM high-energy electrons pass through a thin sample and form a projected image on a screen or image sensor, similar to the way a projector forms a magnified image of a frame of photographic film.

Due to the small size of the electrons, which are easily deflected under the action of hydrocarbons or gas molecules, it is necessary to use the electron beam in a vacuum environment [START_REF] Dubochet | Electron microscopy of frozen water and aqueous solutions[END_REF]. Moreover, by the reason of the incompatibility between the requirements for vacuum and the natural hydrated state of biological samples, it was decided to keep the sample frozen at the temperature of liquid nitrogen or helium throughout the experiment.

Sample preparation and data acquisition

The first steps concern of course the sample purification, dilution to the appropriate concentration in a chosen buffer. The sample must then be deposited on an EM grid (copper and more rarely gold) that is often covered by a carbon layer (Fig. 39). The next step is to freeze the sample in liquid ethane using a device referred to as the "vitrobot". A drop of sample is usually applied on the grid under high humidity and low temperature conditions, blotted to remove the excess of liquid to get monolayer of sample and immediately plunged into liquid ethane cooled down to the temperature of liquid nitrogen. Then the sample is transferred to liquid nitrogen. The point being to freeze the sample in vitreous (non-crystalline) ice. After that, the sample is visualized and collected on a cryo-electron microscope to obtain 2D projections. During data collection and irradiating a sample with electrons, the protein being denser than ice, appears darker on the image, mainly due to phase contrast. At the end, the sample is analyzed using certain programs and an atomic model is built (Fig. 40).

Cryo-electron microscopes

Titan Krios

Titan Krios is the latest generation electron microscope with capacities for high-resolution data collection. The electron source is a Field Emission Gun (FEG) that can be operated at 80keV, 100keV, 200keV or 300keV. The advantages of this microscope are an autoloader mechanism allows mounting twelve grids at a time and 2 nd objective lens. The first data acquisition was performed in Netherlands

Center for Electron Nanoscopy (NeCEN, Leiden) on Titan microscope equipped with Falcon II detector.

Tecnai F30 Polara

The Tecnai F30 Polara cryo-electron microscope allows to collect the data for both cryoelectron tomography and single particle analysis experiments. Its FEG is usually tuned to 100keV, 200keV or 300keV. The grids are manually mounted six at a time. Several data acquisitions were performed on this microscope located in the Centre de Biologie Intégrative (CBI, Illkirch, France).

Talos Arctica

The Talos Arctica is a fully automated 200 kV FEG electron microscope. This microscope is also used for cryo-electron tomography and single particle cryo-electron microscopy. It is equipped with an autoloader system that permits loading of up to twelve specimens. The latest data acquisitions were performed on the Talos Arctica microscope located in European Institute of Chemistry and Biology (IECB, Pessac, France).

Table 7 shows the characteristics of the Titan Krios, Tecnai F30 Polara and Talos Arctica microscopes. -0,6 --4,0

-1,5 --4,5 -0,5 --3,0

Pixel size 1,04 1,81 1,24 Magnification 134000x 59000x 120000x

Image processing

Can be subdivided to several sequential tasks:

Frame re-alignment

With the advent of direct detector device (DDD) cameras, it became possible to collect a cryo-EM image as "movie", i.e.; multiple frames covering few seconds of exposure, which allows potentially for the correction of movement of the specimen, due both to instabilities in the microscope specimen stage and electron beam-induced movements, simply by realigning corresponding areas of the grid from the same movie. Subsequently, the quality of the integrated images improves significantly and can potentially go to atomic resolutions [START_REF] Baker | The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals[END_REF][START_REF] Scheres | Beam-induced motion correction for sub-megadalton cryo-EM particles eLife[END_REF].

Contrast transfer function

It is a mathematical function that describes how aberrations in a TEM modify the image of a sample, expressed in Fourier space. The correction is necessary because the CTF causes resolutiondependent amplitude modulations (Thon rings;[START_REF] Thon | Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung[END_REF] and phase reversals in the image. Without any CTF correction, reconstructed model may contain significant local density displacements. Some parameters, such as the wavelength and the coefficient of spherical aberration, are given by the microscope settings, while others (defocusing and astigmatism) are established experimentally. After mathematical calculation of the СTF, images should be assessed qualitatively for drift, astigmatism, and overall resolution. The CTF is an oscillating function which crosses zero several times. When the function values are 0, information is lost, therefore, it is necessary to collect data at different defocus settings, which leads to averaged values with the CTF correction (Penczek, 2010).

Particle picking and extraction

After selection of good micrographs based partly on the Thon rings, particles are picked manually or semi-automatically and then extracted from the micrographs for further manipulations.

Particle sorting (classification)

Classification is needed in order to obtain homogeneous sets of particle that represent different ensembles of conformations. Classification can be in two or three dimensions (2D or 3D). The 2D classification is often deployed without a prior reference (reference-free) simply by cross-matching all images to each other's, which gives an initial idea of the heterogeneity in the data set. 3D classification on the other hand allows to sort different particle images after reconstruction them in several different 3D volumes (cryo-EM reconstructions). The accuracy of the classification depends largely on the quality of the sample. The image processing algorithms are still limited by the amount of structural heterogeneity that can be tolerated. Classifying small, asymmetric and/or extremely dynamic samples remains significantly challenging.

Refinement

After classification, the stage of optimized refinement begins, which reduces the tendency of the model to correlate noise in one image, thereby increasing the accuracy and quality of the resulting map. This process is iterative and can potentially yield cryo-EM reconstructions at atomic resolutions.

The image processing steps are summarized in the figure 41 below.

Resolution in cryo-EM

Resolution is probably the most important parameter in assessing the quality of an electron

RESULTS

Functional tests of initiation factors

To check the initiation factors activity and the best ratio between factors and ribosome for the complex formation we used toeprinting assay. There are many methods that could be used but most of them either require a large amount of material or use the E. coli system. In the 90s, Larry Gold showed the possibility of checking the activity of initiation factors by toeprinting method.

IF1 activity

As IF1 promotes the binding of the IF2 to the 30S ribosomal subunit [START_REF] Stringer | Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli[END_REF][START_REF] Weiel | The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization[END_REF][START_REF] Celano | Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes[END_REF], it is possible to check the concentration of IF1 with fixed concentration of IF2.

On the first lane on the figure 42, due to the adding mRNA only a signal of full-length extension appeared. One the second lane, mRNA was blocked by 30S that is demonstrated by appearing of several bands more or less at the right position. One the next lanes, adding a different concentration of IF1, the concentrations of mRNA, 30S, fMet-tRNA fMet and IF2 were left the same.

The quantification of the complex formation was done by estimation of amount of signal at position +16 in relation to the sum of +16 signal and the signal of full length in the ImageQuant TL software (Fig. 43).

The best ratio for the complex formation is 3 time more of the IF1 compare to the ribosome.

IF2 activity

IF2 promotes the binding of the fMet-tRNA fMet to the 30S [START_REF] Lockwood | A complex between initiation factor IF2, guanosine triphosphate, and fMet-tRNA: an intermediate in initiation complex formation[END_REF][START_REF] Majumdar | Specific binding of Excherichia coli chain Initiation factor 2 to fMet-tRnafMet[END_REF][START_REF] Sundari | Interaction of bacterial initiation factor 2 with initiator tRNA[END_REF][START_REF] Teana | Late events in translation initiation. Adjustment of fMet-tRNA in the ribosomal P-site[END_REF]. The IF2 activity was checked with the fixed concentration of fMet-tRNA fMet . The protocol we used was the same as for IF1 and the selected concentration corresponded to the concentration of IF1 i.e. 3 times more than ribosome (Fig. 44,45).

IF3 activity

IF3 can specifically recognize only methionine tRNA. In Hartz et al article that was published in 1990, it was shown that when they added IF3, the toe-prints signals of cysteine and phenylalanine tRNAs disappeared, while the methionine tRNA signal was still present.

We used this idea to test the third factor by adding lysine tRNA. In the presence of not fully active IF3 there are signals of different tRNAs but when the IF3 is active, the band that was correspond to Lys-tRNA disappeared. The best concentration of IF3 selected for further experiments was 2 times more that the concentration of 30S (Fig.

46, 47).

The ratio used in the work:

IF1 3:1 IF2 3:1 IF3 2:1 mRNA 3:1 fMet-tRNA fMet 3:1
The first purification of 30S was done from 70S by separation of two subunits in sucrose gradients with low magnesium (1 mM Mg(OAc)2) and high salt (200 mM KCl) buffer concentrations. The 30S preparation was checked on agarose gel in denaturing conditions. No degradation was observed (Fig. 48). The ratio of the components was taken from the literature.

Mass spectrometry

Analysis of the small ribosomal subunit shows the presence of all proteins, including S1 protein with a low number of spectra count. This result in combination with other studies of S1 protein probably indicates that S1 from S. aureus is not a ribosomal protein. Only one nonribosomal protein was found in the sample with 1 spectra count (Table 8).

Negative stain electron microscopy

Negative stain electron microscopy (EM) is the best method for rapid sample assessment using the contrast enhancing stain reagent. This technique is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy.

The disadvantage of negative stain EM is the limited resolution up to 18 -20 Å.

This approach can be used to verify the sample concentration required to the grids preparation for cryo-EM. The process itself consists in adsorbing the sample on a substrate, then a stain is applied, blotted and dried to obtain a thin layer of electron dense stain in which the particles are embedded (Fig. 49). All procedures were performed by Dr. Yaser Hashem.

st data acquisition

All procedures related to data collection or image processing were performed in Dr.

Yaser Hashem laboratory by engineer Dr. Heddy Soufari. 4 μl of 90 nM S. aureus initiation complex was applied to the cryo-EM grid, blotted with filter paper from both sides for 3 One class was obtained with a moving head and an unstable structure. Also, there were several classes with flexible helix 44 (Fig. 50).

The effect of salt concentration on the 30S purification was checked by the sucrose gradient and then all samples were loaded on the 15% PAAG (Fig. 51, 52). Results reveal that under high salt conditions (namely 200 mM KCl or 400 mM NaCl) some of the proteins of small ribosomal subunit dissociate from the ribosome. We suggest that it is bS2 (upper band) and another unidentified protein (lower band) (Fig. 52).

For large-scale purification conditions 100 mM NH4Cl were chosen as the most optimal.

Also, an additional gradient was added to completely remove 50S from the sample.

Then, 30S preparation was checked on agarose gel in denaturing conditions to detect rRNA degradation (Fig. 53). A B

nd data acquisition

The sample with the correct ratio in low salt conditions was prepared for data collection on Polara microscope followed by data processing on Scipion.

The structure of 30S with mRNA, fMet-tRNA fMet and IF2 was obtained at 10 Å. The head of the 30S was not so flexible, at the same time the helix 44 was still in two positions in around 86% of the particles (Fig. 54).

Perhaps the stage of ribosome purification was too long, it was decided to purify the small ribosomal subunit without additional sucrose gradient and do not freeze the sample before loading on the grids. The 30S preparation was checked on agarose gel in denaturing conditions (Fig. 55).

rd data acquisition

Two 30S with tRNA and IF3 structures in two conformations and one structure with IF2

were obtained, but still the helix 44 was flexible in around 60% of particles (Fig. 56).

Further, it was decided to optimize the ribosome purification step again. In 1977, it was shown the ribosomal subunits separation in 30 mM NH4Cl buffer [START_REF] Fahnestock | Reconstitution of active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis[END_REF]. Further experiments were performed in low salt buffer conditions. In 10 mM Mg(OAc)2 and low salt conditions the 70S was stable. In 1 mM Mg(OAc)2 subunits separation became possible while in 2 mM Mg(OAc)2 only partial separation was observed (Fig. 57).

For large-scale purification conditions 30 mM NH4Cl and 1 mM Mg(OAc)2 were chosen as the most optimal.

Stepwise initiation complex assembling

According to Marina Rodnina's and Venki Ramakrishnan's articles we designed the experiments progression that is summarized in the scheme below.

Inspired by crystallography it was decided to add spermidine to stabilize rRNA.

All planned experiments presented in Scheme 58 below. Scheme of stepwise assembling of initiation complex

th data acquisition

After several hours of data collection on Talos microscope, the preliminary structure of the small ribosomal subunit (30S) was obtained at 10 Å. In five classes the flexibility of helix 44 was still observed (Fig. 59).

The flexible h44 was stabilized by adding spermidine at 2,5 mM concentration. The structure was processed in one class, no rRNA flexibility was observed (Fig. 60).

th data acquisition

The next step was the addition of spa mRNA to the previous conditions. Different head positions were observed with (Fig. 62) and without spermidine (Fig. 61). The 30S structure with spermidine in closed conformation was obtained at 4.39 Å after 2 hours of data collection on Talos microscope. Data was processed in one class with the CryoSparc software. (Fig. 63), 2 nd sample was prepared by incubating 30S with mRNA at 37 o C for 10 minutes and then with IF1 and IF3 for additional 5 minutes (340416 particles) (Fig. 64). rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red and IF3 is in pink color (5LI0). IF1 was not detected.

A B

th data acquisition

Next, it was decided to add stepwise the remaining components of the initiation complex. Variations of the prepared complexes are described below in the Table 9. Initially, 30S with mRNA was incubated at 37 o C for 5 min and then the rest components were added without incubation as described in Hussain et al and Milon et al articles.

Table 9. Variations of prepared complexes collected on Talos microscope and processed with Cryosparc software.

1 complex 2 complex 3 complex 4 complex 5 complex

30S + + + + + mRNA + + + + + IF1 + + + IF2 + + + + IF3 + + + + + fMet-tRNA fMet + + +
Unfortunately, the presence of 1 and 2 factors was not detected in any complex while IF3 was found in each complex (Fig. 65, 66, 67, 68 and 69).

Figure 65. Structure obtained after 6th data collection: 30S with mRNA, fMet-tRNA fMet and IF3 complex reconstruction (A) with the close-up view on mRNA (B). IF1 was not detected (complex 1). rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red, IF3 is in pink and fMet-tRNA fMet is in green colors (5LI0, 124344 particles). The estimated resolution in CryoSparc was 6.8 Å (C).

A B C

Figure 66. Cryo-EM reconstruction of 30S with mRNA and IF3 complex (A) with a close-up view on mRNA (B) at 8.5 Å (C). IF2 was not detected (complex 2). rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red and IF3 is in pink colors (5LI0, 107292 particles).

A B C

Figure 67. Structure obtained after 6th data collection: 30S with mRNA, fMet-tRNA fMet and IF3 (A) with closeup view on mRNA (B). IF2 was not detected (complex 3). rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red, IF3 is in pink and fMet-tRNA fMet is in green colors (5LI0, 90598 particles). The resolution was calculated in CryoSparc software (C).

A B C

Figure 68. Structure obtained after 6th data collection: 30S with mRNA and IF3 (A) with close-up view on mRNA (B) at 4.7 Å (C). IF1 and IF2 were not detected (complex 4). rRNA is highlighted in yellow, rproteins are in orange, mRNA is in red and IF3 is in pink colors (5LI0, 136627 particles).

A B C

All obtained results are summarized and presented in Scheme 70.

A B C

Scheme of stepwise assembling of initiation complex To obtain the initiation complex close to natural conditions, there is a method whose idea is to prevent the binding of subunits by decreasing the magnesium concentration with a non-hydrolysable GTP analog. Non-hydrolysable analogues of GTP, that irreversibly activates G proteins, are guanyl-5'-imidodiphosphate (GMPPNP) and 5'-guanosinemethylene-triphosphate (GMPPCP). GMPPNP and GMPPCP are synthetically prepared analogs where one of the oxygen atoms is replaced with amine or methylene groups. These components perform the functions of preventing GTP hydrolysis and large subunit joining to the initiation complex, as well as stalling of GTP-dependent complexes. This method has been used on mammalian cells and on kinetoplastids [START_REF] Anthony | Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition[END_REF][START_REF] Hershey | A competitive inhibitor of the GTP reaction in protein synthesis[END_REF][START_REF] Gray | Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs[END_REF][START_REF] Simonetti | eIF3 Peripheral Subunits Rearrangement after mRNA Binding and Start-Codon Recognition[END_REF]. A similar experiment with some adaptations for S. aureus is described below.

The S. aureus cell extract treated or not with GMP-PNP were fractionated by sucrose density gradients (Fig. 71) and then the obtained fractions were centrifuged in order to concentrate the sample. In order to do not inhibit the dissociation by spermidine (Umekage and Ueda, 2006) its concentration was reduced to 0.25 mM. Further, the sample was resuspended in buffer with 30 mM NH4Cl, 5 mM Mg(OAc)2 and 10 mM Hepes-KOH pH 7.5

and then analyzed on SDS-PAGE (Fig. 72) and on native agarose gel (Fig. 73).

Ex vivo experiments 7 th data acquisition

Then the sample was collected on Talos microscope and processed with Relion software.

Unfortunately, all the particles had a flexible helix 44 (Fig. 74).

DISCUSSION

Protein synthesis is a fundamental process found in all living cells. Despite very high conservation of this processes within bacteria, many species-specific features in the structures of ribosomes and translation factors in different species exist. In our studies, we focused on initiation step as it is a fundamental rate limiting and tightly controlled stage and is a target of various regulatory mechanisms.

According to recent studies, significant differences between gram-positive (S. aureus and B. subtilis) and gram-negative (T. thermophilus and E. coli) bacteria were observed. These peculiarities are primarily related to the ribosome structure. One of the most interesting examples is the S. aureus ribosome and its extension of helix 26 that binds close to Shine-Dalgarno (SD) region and its involvement in forming 100S inactive dimers. Dimers are formed in response to stressful conditions for the protection and storage state. The process itself is called the ribosome hibernation. In S. aureus, it was shown that only one long HPF (long hibernation promoting factor) protein is involved in this process, while in E. coli bacteria three proteins are involved in this process: YfiA, RMF (ribosome modulation factor) and short HPF [START_REF] Khusainov | Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF[END_REF][START_REF] Polikanov | How hibernation factors RMF, HPF, and YfiA turn off protein synthesis[END_REF]. Another feature of S. aureus ribosomes is the absence of S1 protein, which in gram-negative bacteria unfolds the highly structured mRNA for the proper accommodation on the 30S ribosomal subunit during the translation initiation stage [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF][START_REF] Duval | Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation[END_REF]. S1 protein is the largest and the most acidic ribosomal protein. In S. aureus, S1 loses the domains responsible for binding to the ribosome.

Using different protocols of ribosome purification in high and low salt conditions, S1 protein was not detected in any case. In addition, this protein was also absent in the structure of S.

aureus 100S dimmers, in contrast to gram-negative bacteria [START_REF] Beckert | Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1[END_REF].

Toeprinting assay was performed in order to obtain the maximum amount of initiation complex and to choose more accurately the IF3 concentration, which causes dissociation of fMet-tRNA fMet from the initiation complex [START_REF] Milon | Transient kinetics, fluorescence and FRET in studies of initiation of translation in bacteria[END_REF]. Toeprinting method was chosen because it requires a very small amount of sample obtained from S. aureus. Due to the need to use E. coli components, "in vitro translation" method was less preferred. The other methods like isothermal titration calorimetry and filter-binding assay require a lot of material and in the case of the second one require also a radioactive tRNAs. Initiation factor 1 activity was checked together with initiation factor 2 (IF2) as it promotes the binding of IF2 to the 30S [START_REF] Stringer | Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli[END_REF][START_REF] Weiel | The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization[END_REF][START_REF] Celano | Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes[END_REF]. It was shown that IF2

promotes the binding of fMet-tRNA fmet to the 30S [START_REF] Lockwood | A complex between initiation factor IF2, guanosine triphosphate, and fMet-tRNA: an intermediate in initiation complex formation[END_REF][START_REF] Majumdar | Specific binding of Excherichia coli chain Initiation factor 2 to fMet-tRnafMet[END_REF][START_REF] Sundari | Interaction of bacterial initiation factor 2 with initiator tRNA[END_REF][START_REF] Teana | Late events in translation initiation. Adjustment of fMet-tRNA in the ribosomal P-site[END_REF]. Its activity was tested by increasing IF2 concentration with fixed concentration of fMet-tRNA fmet . The optimal ratio of IF1 and IF2 to the 30S was found and corresponds to 3:1 proteins concentration to the ribosome concentration. The idea of checking the initiation factor 3 (IF3) activity by toe-printing assay was taken from the article Hartz et al., that was published in 1990. For their experiments, they used 3 types of tRNAs (methionine, cysteine and phenylalanine). They observed that IF3 has a specific function in the recognition of the methionine tRNA. It was shown that with the fully active IF3 the toe-print signals of other tRNAs disappeared. The same kind of experiment was performed and the best concentration of IF3 was found to be twice the ribosome concentration. The obtained results correspond to the literature data used for the initiation complex formation [START_REF] Simonetti | Structure of the 30S translation initiation complex[END_REF].

The negative stain electron microscopy (EM) was performed to find the optimal sample concentration for cryo-EM. Then the initiation complex was prepared and applied to the cryo-EM grids. The data was collected on the Titan microscope and processed in Scipion software package that includes Relion application for the 2D and 3D classification. As a result of data processing, one class with invisible 30S head was observed and in other classes the helix 44 was very flexible. After the salt conditions screening, it was possible to slightly increase the number of particles with proper conformation of the helix 44. The complex of the 30S with mRNA, fMet-tRNA fmet and IF2 was also obtained at 10 Å. However, in most cases the structure was unstable. The next data acquisition was performed with the fresh 30S preparation, which again slightly improved the number of good particles. Three low-resolution structures were obtained with IF2 and IF3 in two different conformations. Further changes in the protocol such as low salt concentration and addition of polyamines led us to obtain the ribosome with a stable conformation of the helix 44. It is known that polyamines or Mg 2+ ions are neutralizing the negative charge of the phosphate-sugar backbone, thus increasing the structural stability (Tabor, 1962, Sakai and[START_REF] Sakai | Regulation of ornithine decarboxylase activity by guanine nucleotides: in vivo test in potassium-depleted Escherichia coli[END_REF]. The binding sites of putrescine with a small ribosomal subunit were shown close to helix 44 (Noeske et al., 2015, Fig. 75). It can be assumed that spermidine may replace it and bind on the same place. In our later attempts, after we have started supplementing our complexes with spermidine in order to stabilize the rRNA, only the structure with the IF3 was obtained, IF1 and IF2 were absent in the structure. This can be explained by the addition of spermidine that may impact the IFs binding. Another hypothesis is the requirement of an additional factor/component for the proper complex formation. For this purpose, the experimental strategy was changed to the method closer to natural conditions. It was found that the effect of the ribosome separation into subunits by GMP-PNP depends on the magnesium concentration. By doubling the concentration of magnesium, the reaction was completely inhibited. Also, by adding additional mRNA double peak occurs, which may indicate the appearance of 30S complexes. This effect is still not clear. Additional experiments are required.

At high concentrations of spermidine, like 2 mM, the separation of ribosomal subunits can be inhibited [START_REF] Umekage | Spermidine inhibits transient and stable ribosome subunit dissociation[END_REF]. Thus, for the aforementioned ex vivo experiments, the spermidine concentration was 10 times less than was used before.

Unfortunately, the obtained structures of 30S demonstrate the need to select concentration of polyamines more accurately.

The effect of the incubation time of the complex on its formation was also investigated.

It was found that the stepwise complex assembly with the minimal incubation of factors is more preferable (Fig. 78).

Comparing the two structures of 30S-mRNA with and without IF3, no difference in 30Shead conformation were observed (Fig. 79).

MATERIALS AND METHODS

80S PURIFICATION

Purification, crystallization and post crystallization treatment of 80S from C. albicans were performed according to a similar protocol described for S. cerevisiae with some adaptations (Ben-Shem et al., 2010).

Buffers were used during the experiment:

Buffer M:

Cell growth

The C. albicans SC5314 strain is a wild type strain isolated from a patient with a generalized infection. It was provided by Dr. Sadri Znaidi (Institut Pasteur, Paris, France) [START_REF] Gillum | Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations[END_REF].

All manipulations with the cells were performed in a specially equipped laboratory with a safety level of 2. Cells were grown in a standard Yeast Extract Peptone Dextrose Agar (YPD) medium at 30 o C till OD600 = 1 and further subjected to glucose starvation. This step is needed to homogenize the ribosomes and reversibly induce translation inhibition. After cells centrifugation, the pellet was resuspended in buffer M.

Lysis 5 grams of cells obtained from 4 liters of culture were resuspended in buffer M with protease inhibitor cocktail (Roche), RNasin (Promega), Pefabloc (Roche), heparin (Sigma-Aldrich) and DTT. Heparin acts on the solubility of ribosomes which affects the amount of PEG used in subsequent steps. The cells were lysed by vortexing with glass beads and were centrifuged at 13000 rpm for 2 min and then 16000 rpm for 9 min using JA 25.50 rotor.

PEG precipitation

PEG20K 30% w/v (Hampton research) was added to the supernatant to final concentration 4.5% w/v and then was centrifuged at 20000 g for 5 minutes to precipitate anything heavier than the ribosome. The concentration of KCl was adjusted till 130 mM. The PEG concentration was increased till 8.5% w/v and was centrifuged at 20000 g for 10 minutes to precipitate ribosomes. Then, the concentration of KCl in buffer M was changed to 150 mM and protease inhibitors, DTT and heparin were additionally added.

Sucrose density gradient centrifugation

The ribosomes were loaded on a 10-30% w/v sucrose gradients and was centrifuged at 18000 rpm for 15 hours using Beckman SW28 swinging rotor (Fig. 80). The appropriate fractions were pooled and concentrations of MgCl2 and KCl were adjusted to 10 mM and 150 mM respectively.

Last PEG precipitation

The PEG20K was added till the final concentration 6.9% w/v to precipitate ribosomes and the sample was centrifuged at 20000 g for 10 min. The pellet was gently resuspended in buffer G to obtain the final concentration 23 mg/ml. The sample was frozen in liquid nitrogen and stored at -80 o C.

X-RAY CRYSTALLOGRAPHY

The other technique I would like to present is X-ray crystallography, because of its relevance to a part of my thesis work. X-ray crystallography is a method that allows the structural determination of macromolecular complexes.

Wilhelm Röntgen discovered the X-ray in 1895. In 1912, Paul Peter Ewald and Max von Laue had the idea that crystals could be used as a diffraction grating for X-rays. Later, Max von Laue proposed a law that relates the scattering angles and the size and orientation of the distances between unit cells in a crystal, for which he was awarded the Nobel Prize in Physics in 1914.

In the 1980s, the large ribosomal subunit (LSU) was first crystallized by the Ada Yonath's in collaboration with Gunter Witmann [START_REF] Yonath | Crystallization of the large ribosomal subunit from B. stearothermophilus[END_REF]. The high-resolution structure of 50S was obtained by Nenad Ban in Thomas Steiz laboratory [START_REF] Ban | The complete atomic structure of the large ribosomal subunit at 2.4 A resolution[END_REF].

The first structures of the complete ribosome, small ribosomal subunit (SSU) and its functional complexes form Thermus thermophilus were obtained by Marat Yusupov and Gulnara Yusupova in the laboratory of Harry Noller and in the laboratory of Venkatraman Ramakrishnan [START_REF] Clemons | Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution[END_REF], Yusupov et al., 2001, Yusupova et al., 2001). The structure of the ribosome with a resolution of 3.5 Å from Escherichia coli was determined in the laboratory of Jamie Cate [START_REF] Schuwirth | Structures of the bacterial ribosome at 3.5 A resolution[END_REF].

Crystallization

The process of transition from liquid to solid crystalline state is called crystallization.

The solid materials obtained during this process are called crystals. The crystallization process is characterized by the nucleation of the crystallite with its subsequent growth to a diffractionquality crystal. The conditions under which the crystal(s) appeared did not always coincide with the conditions for their growth. The crystallographer's goal is to find those conditions under which one crystal with maximum size grows, since such crystals better diffract.

Figure 81 illustrates a phase diagram where solubility varies depending on the concentration of precipitant (e.g., polyethylene glycol (PEG) or salt). In the undersaturated region the crystals dissolve, while in the supersaturated region (metastable zone and labile zone) they grow. In practice, for the crystal formation, the protein concentration should exceed the solubility by at least three times. This is necessary to overcome the energy barrier that exists during crystal formation and represents the free energy needed to create a small cluster of proteins. In the "metastable zone", the supersaturation is too small, the nucleation rate is low, and the crystals formation takes a sufficiently long period of time. In the adjacent "labile zone", the supersaturation is large enough and spontaneous nucleation is observed. In the "precipitation zone" due to excessive supersaturation occurs aggregate formation and precipitation [START_REF] Asherie | Protein crystallization and phase diagrams[END_REF].

Many factors can influence the appearance and growth of crystals. Initially, a large search is carried out for optimal conditions with changes in physical and chemical parameters like changes in sample concentration, buffer components, pH, concentration of the precipitant and temperature conditions. All these manipulations can be carried out both manually and on specially designed robots.

Overview of single-crystal X-ray diffraction

This technique has three main steps. The first step is to obtain sufficiently large crystals that are pure in composition and regular in structure. The second step is to place the crystal in an X-ray beam, producing a regular pattern of reflections. The intensities and directions of the diffracted X-rays are simultaneously recorded by the diffractometer. At the third stage, special algorithms are used to produce and refine the model of the arrangement of atoms within the crystal. The refined structure is called the crystal structure and is usually stored in a public database (Fig. 82).

Resolution in X-ray crystallography

Resolution in X-ray crystallography is a physical parameter and is the distance from the center till the spot on the diffraction pattern. It is a more accurate parameter compared to electron microscopy.

Analytical ultracentrifugation

Analytical ultracentrifugation is a method for the quantitative analysis of macromolecules in solution. The optical systems (absorbance and fluorescence) permit precise and selective observation of sedimentation in real time [START_REF] Cole | Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium[END_REF].

The experiments were performed at 4 o C using Beckman XL-I analytical ultracentrifuge and Beckman An-50Ti rotor. Thawed ribosomes were diluted to 1 OD/ml (260 nm) and was loaded into quartz cuvette. Scans were made every 4 min at 260 nm and 280 nm. The data analysis was done in Sednterp and Sedfit software.

After freezing the major part of the sample stays in 80S form. Also, a small amount of dimers is present in the sample (Fig. 85).

Crystallization

The initial conditions for crystallization were taken from the David Bruchlen's manuscript and were adapted over time to produce reproducible crystals. First screenings were carried out by the robot (Formulatrix, CBI, IGBMC) and then manually at 4 o C. All tried and tested conditions are summarized in Table 11 below. Other changes in crystallization conditions:

• ribosome concentration,

• volume of reservoir solution,

• ratio in the drop between sample and reservoir,

• hanging and sitting drops,

• sample incubation at 30 o C and at 37 o C

• crystallization temperature 4 o C and 20 o C.

The first crystal screenings on the robot did not lead to large crystals, so it was decided to change the crystallization parameters manually. It was found that the concentration of KSCN significantly affects the crystal shape and the volume of reservoir affects the size of crystal. Also, decrease of ribosome and spermidine concentrations directly affected on the crystal shape. As a result, the obtained crystals diffracted at 7 Å (Fig. 86).

Post-crystallization treatment

This stage is needed to improve the crystal stability and diffraction. For this purpose, the dehydration method was used. It is known that the reduction of the solvent content inside the crystal produces internal strengthening which can lead to an improvement in the diffraction limit. To achieve this goal, our laboratory uses a slow and stepwise increase of smaller PEG than 20K, depending on the type of which the cell parameter and the degree of conformational changes of the small ribosomal subunit can vary.

The post-crystallization treatment conditions were taken from David Bruchlen's manuscript to check the quality of crystals. Initially, the protocol was developed for Saccharomyces cerevisiae which was later adapted for cryoprotection of Candida albicans's crystals.

The drop on cover slip was placed into a small Petri dish and the first buffer was replaced by the solution with slightly higher PEG 20K concentration (80 mM Bis-tris-Ac pH 7.0, 70 mM KSCN, 10 mM Mg(OAc) 2 , 20% v/v Glycerol, 5% w/v PEG 20K, 6.5 mM spermidine, 7.5 mM NH4OAc, 1.4 mM Deoxy Big Chap, 2 mM DTT). Then this solution was replaced

DISCUSSION

Most antibiotics inhibit protein synthesis by binding to the ribosome, so it is necessary to know the ribosome structure for the correct understanding the mechanism of action of these compounds.

Candida albicans is a pathogenic yeast belonging to the Saccharomycetaceae family.

The previous biochemical, structural and bioinformatics studies show a high level of structural similarity between 80S from S. cerevisiae and C. albicans (see Bruchlen's manuscript). In this regard, it was decided to use the cell growth and ribosome purification protocols developed for S. cerevisiae with some adaptations. After obtaining the good quality sample for 80S crystallization the protocol from S. cerevisiae were used [START_REF] Ben-Shem | The structure of the eukaryotic ribosome at 3.0 A resolution[END_REF]. However, the crystallization assays were unsuccessful using the related protocols from yeast, as no crystals were found in our drops. A large screening was carried out to determine the conditions for the appearance and growth of crystals. Subsequently, the crystallization conditions were changed to obtain better reproducibility. Several conditions were found under which the crystals diffracted at 6,5 -7 Å.

It should be noted that the only successfully crystallized yeast ribosomes are vacant and do not contain any translation factors. However, these ribosomes are further stabilized by a non-ribosomal protein, a homologue of stm1, which occupies the mRNA tunnel in order to inhibit translation during the stress by glucose starvation [START_REF] Ben-Shem | The structure of the eukaryotic ribosome at 3.0 A resolution[END_REF]. This is another significant similarity between S. cerevisiae and C. albicans ribosomes.

PERSPECTIVES

Candida albicans (C. albicans) is detected in the gastrointestinal tract and mouth in 40-60% of healthy adults [START_REF] Kerawala | Oral and maxillofacial surgery[END_REF], but despite this, mortality among patients with systemic candidiasis reaches 40% [START_REF] Singh | Invasive Candidiasis in the Southeast-Asian Region[END_REF]. Invasive candidiasis contracted in a hospital causes 2800 to 11200 deaths yearly in the USA [START_REF] Pfaller | Epidemiology of Invasive Candidiasis: A Persistent Public Health Problem[END_REF]. Nevertheless, these numbers may not truly reflect the true extent of damage this organism causes, given new studies indicating that C. albicans can cross the blood-brain barrier [START_REF] Wu | Microglia and amyloid precursor protein coordinate control of transient Candida cerebritis with memory deficits[END_REF]. Thus, there is a need to search for compounds effective against this pathogenic yeast.

Since most antibiotic targets are ribosomes, determining their structure is essential. After obtaining well-reproducible protocols for ribosome purification and crystallization, the next step is to improve diffraction of crystals.

At the same time, the predicted С. albicans ribosome structure allowed our collaborators to begin the development of species-specific compounds, which may be further used in in vivo studies. Before reaching the ribosome, the compounds must pass through the cell wall and plasma membrane. If at least one of the ribosomal inhibitors stops the growth of the strain, then it will be important to verify the non-toxicity of this molecule on human cells. This is a prerequisite for obtaining a new drug candidate, which will be a specific antifungal against C.

albicans.

CONCLUDING REMARKS

The structural studies of ribosome and its functional complexes are necessary and have a practical significance. Scientists from around the world are contributing to this challenge.

They face the fact that not all ribosome structures have been resolved yet and, when working in this direction, they reveal many unexplored and essential features that require more detailed study. After stepwise solving every task, new important challenges arise.

The reproducible protocols are the small but significant step in ribosome structure determination from C. albicans. This structure will be further used to study the binding of new compounds to the C. albicans ribosome. To solve this problem, X-ray crystallography is the best suited. Bioinformatics data already allows our collaborators to begin the selection of new compounds specifically targets C. albicans ribosome, which will soon be used in our research. La structure du ribosome résolue il y a trois ans présente des caractéristiques spécifiques qui ne sont pas typiques des bactéries à gram négatif [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF]. L'une des caractéristiques de l'ARN ribosomal dans la petite sous-unité ribosomal est l'extension de l'hélice 26, impliquée dans la dimérisation des ribosomes [START_REF] Khusainov | Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF[END_REF]. Près de 123 cette hélice se lie la protéine ribosomale S1 qui, chez E. coli, déploie l'ARNm structuré sur le ribosome. Cependant, il a été démontré que Bacillus stearothermophilus (B.

RESUME DE THESE EN FRANÇAIS

stearothermophilus) est dépourvu de protéine équivalente à E. coli S1 [START_REF] Isono | Role of ribosomal protein S1 in portein synthesis: effects of its addition to Bacillus stearothermophilus cell-free system[END_REF].

Étant donné que S1 est très similaire chez toutes les bactéries à Gram positif et que chez S.

aureus le site de liaison aux ribosomes a été perdu, il a été suggéré que les facteurs d'initiation (IF) pourraient probablement remplir des fonctions supplémentaires compensant le rôle de S1.

De plus, les facteurs d'initiation ont aussi des caractéristiques spécifiques à l'espèce. La région C-terminale de IF2 est très conservée, tandis que la longueur de la région N-terminale dépend de l'espèce bactérienne.

En outre, chez S. aureus, il existe des ARN régulateurs, tels que l'ARN III, qui affectent la régulation des gènes de virulence. Il a été montré que l'ARN III réprime l'expression du gène spa qui code pour la protéine de surface A, l'un des principaux facteurs de virulence, et inhibe la formation du complexe d'initiation de la traduction [START_REF] Patel | Virulence of protein Adeficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement[END_REF][START_REF] Huntzinger | Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression[END_REF].

L'objectif principal de notre projet était de déterminer les structures par cryo-EM des complexes d'initiation de Staphylococcus aureus, afin de mieux comprendre certains aspects fondamentaux et spécifiques de l'initiation de la traduction de ce pathogène.

Projet de recherche 2

Candida albicans (С. albicans) est l'agent pathogène humain le plus répandu dans au moins 70% de la population humaine. Du fait que les cibles cellulaires de C. albicans sont souvent partagées avec les humains, de nombreux effets secondaires apparaissent, rendant le traitement inefficace [START_REF] Berman | Candida albicans: a molecular revolution built on lessons from budding yeast[END_REF]. Par conséquent, l'étude des mécanismes physiologiques de cet agent pathogène et la mise au point de nouveaux médicaments contre celui-ci ont une signification pratique.

Le ribosome est la cible des antibiotiques les plus couramment utilisés. La détermination de sa structure est une tâche prioritaire.

Les prédictions bio-informatiques montrent une différence significative dans le site E entre C. albicans et S. cerevisiae, ce qui pourrait expliquer la résistance du premier au cycloheximide [START_REF] Goldway | Multidrug resistance in Candida albicans: disruption of the BENr gene[END_REF].

Mon objectif dans ce projet était d'optimiser les protocoles de purification et de cristallisation du ribosome C. albicans et de les rendre reproductibles. Une amélioration supplémentaire de la résolution de la structure ribosomale est nécessaire pour étudier l'effet des antibiotiques.

Résultats

Etude en microscopie cryo-electronique du complexe d'initiation de la traduction de S. aureus

Notre travail comportait plusieurs étapes:

• pour la reconstruction in vitro La stratégie expérimentale a été modifiée en faveur d'une méthode plus proche des conditions naturelles. Il a été démontré que les ribosomes eucaryotes peuvent être séparés en sous-unités par addition de GMP-PNP [START_REF] Anthony | Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition[END_REF][START_REF] Simonetti | eIF3 Peripheral Subunits Rearrangement after mRNA Binding and Start-Codon Recognition[END_REF]. Le même protocole avec quelques adaptations a été utilisé dans notre cas. Au cours de l'analyse des données, il a été observé que, dans toutes les structures obtenues, l'hélice 44 était flexible, probablement en raison de la faible concentration de spermidine. Les résultats montrent la nécessité de sélectionner plus précisément la concentration de spermidine.

L'effet de l'incubation par étapes sur la formation du complexe a également été vérifié.

Il a été constaté que l'incubation séparée de 30S uniquement avec l'ARNm et l'addition de facteurs juste avant la préparation de la grille permettait d'obtenir des structures avec un pourcentage plus élevé d'IF3 lié au ribosome.

Les résultats que j'ai obtenus au cours de ma thèse seront discutés en détail dans les prochains chapitres.

Figure 3 .Figure 12 .Figure 15 .Figure 22 .

 3121522 Figure 3. Composition of bacterial and eukaryotic ribosomes Figure 4. Ribosomal proteins of the 40S subunit Figure 5. Ribosomal proteins of the 60S subunit Figure 6. Sequence and secondary structure of rRNA of LSU from S. cerevisiae Figure 7. Sequence and secondary structure of rRNA of 18S from S. cerevisiae Figure 8. Scheme of the initiation process in bacteria Figure 9. Translation initiation process in eukaryotes Figure 10. Conservative elongation process of protein synthesis in all kingdoms Figure 11. The diagram shows the last 2 steps of translation termination and recycling for prokaryotes Figure 12. The diagram shows termination and recycling processes for eukaryotes that are different from prokaryotes Figure 13. Schematic diagram of translation cycle in bacteria Figure 14. The translation cycle for eukaryotes which is divided into four main steps: initiation (including preinitiation), elongation, termination and recycling Figure 15. The process of the initiation complex formation based on FRET experiments Figure 16. The assembly of the initiation complex, based on cryo-EM structures Figure 17. The structure of IF1 from S. aureus determined with multidimensional NMR spectroscopy Figure 18. X-ray structure of translation IF2 from Methanothermobacter thermautotrophicus Figure 19. Cryo-EM structure of IF3 from Thermus thermophilus Figure 20. Comparison of initiator and elongator methionine tRNAs Figure 21. RNA-mediated mechanism used in virulence gene regulation. The mechanism of action of S. aureus RNAIII on spa expression Figure 22. Scheme of the initiation factor 2 structure from T. thermophilus, S. aureus and E.coli

Figure 23 .Figure 24 .

 2324 Figure 23. A scheme shows at which stage of protein synthesis different antibiotics bind to the ribosome Figure 24. Sucrose gradient profile of 70S from S. aureus Figure 25. One-dimensional PAGE of S. aureus 70S ribosome Figure 26. Agarose gel in native and denaturing conditions Figure 27. Crystals of 70S ribosome from S. aureus Figure 28. Sucrose gradient profile of 30S from S. aureus Figure 29. One-dimensional PAGE of S. aureus 30S ribosome Figure 30. Agarose gel of 30S ribosome Figure 31. Formation of simplified translation initiation complex by toeprinting assays Figure 32. One-dimensional PAGE of initiation factors Figure 33. Structure of spa mRNA Figure 34. 6% PAGE of spa mRNA

Figure 35 .Figure 44 .Figure 46 .Figure 47 .Figure 55 .Figure 57 .Figure 68 .Figure 70 .

 3544464755576870 Figure 35. One-dimensional PAGE of MetRS Figure 36. One-dimensional PAGE of MTF Figure 37. 6% PAGE of itRNA Figure 38. Schematic representation of basic composition of electron microscope Figure 39. Representation of the cryo-EM grid at different scales Figure 40. Scheme of sample analysis by cryo-electron microscopy Figure 41. Principle steps of image processing in CryoSparc Figure 42. Formation of simplified initiation complex with IF1 and IF2 by toeprinting assays Figure 43. The quantification of the toeprint (given in %) was first normalized according to the full-length extension product bands using the ImageQuant TL software Figure 44. Formation of simplified initiation complex with IF2 by toeprinting assays Figure 45. The quantification of the toeprint (given in %) was first normalized according to the full-length extension product bands using the ImageQuant TL software Figure 46. Formation of initiation complex with fMet-tRNA fMet and Lys-tRNA by toeprinting assays Figure 47. The quantification of the toeprint (given in %) was first normalized according to the full-length extension product bands using the ImageQuant TL software Figure 48. Sucrose gradient profile and agarose gel of 30S Figure 49. Micrograph of SSU from S. aureus Figure 50. 3D classification of 643532 particles divided into 10 classes Figure 51. Sucrose gradient profiles of 30S Figure 52. 15% polyacrylamide gel Figure 53. Sucrose gradient profile and agarose gel of 16S rRNA Figure 54. 3D classification of 479872 particles divided into 10 classes. Obtained 1 class with 60000 particles was reclassified into 6 classes Figure 55. Sucrose gradient profile and agarose gel of 16S rRNA Figure 56. During 3D classification 1 class with 40000 particles was obtained and reclassified into 4 classes Figure 57. Sucrose gradient profiles Figure 58. Scheme of stepwise assembling of initiation complex Figure 59. Structures obtained after 4 th data collection: 30S without spermidine Figure 60. Structure obtained after 4 th data collection: 30S with spermidine Figure 61. Structure obtained after 5 th data collection Figure 62. Cryo-EM reconstruction of 30S, mRNA and spermidine complex Figure 63. Structure obtained after 6 th data acquisition Figure 64. Structure obtained after 6 th data acquisition Figure 65. Structure obtained after 6 th data acquisition Figure 66. Cryo-EM reconstruction of 30S with mRNA and IF3 complex Figure 67. Structure obtained after 6 th data collection: 30S with mRNA, fMet-tRNA fMet and IF3 Figure 68. Structure obtained after 6 th data collection: 30S with mRNA and IF3 Figure 69. Structure obtained after 6 th data collection: 30S with mRNA, fMet-tRNA fMet and IF3 Figure 70. Scheme of stepwise assembling of initiation complex Figure 71. Sucrose gradient profile

Figure 72 .Figure 82 .Figure 86 .

 728286 Figure 72. 15% polyacrylamide gel 97 Figure 73. Agarose gel in native conditions of sample from III gradient 97 Figure 74. Structure obtained after 7 th data collection 98 Figure 75. Atomic model of E. coli ribosome (Noeske et al., 2015) Figure 76. Comparison of two 30S structures: without and with spermidine Figure 77. Comparison of two 30S with mRNA structures: without and with spermidine Figure 78. Comparison of two 30S with mRNA and IF3 (different incubation) Figure 79. Comparison of two 30S-mRNA structures: without and with IF3 Figure 80. Sucrose gradient profile of C. albicans ribosome Figure 81. A schematic phase diagram showing the solubility of a protein in solution as a function of the concentration of the precipitant present Figure 82. Schematic representation of diffraction experiment Figure 83. One-dimensional 15% PAGE of 80S from C. albicans and S. cerevisiae Figure 84. Agarose gel of 80S from C. albicans and S. cerevisiae Figure 85. Comparison of sedimentation profiles of C. albicans ribosome and S. cerevisiae ribosome Figure 86. Crystals of 80S from C. albicans Figure 87. Scheme of post-crystallization treatment

For

  the first time, ribosomes were observed by cell biologist George Emil Palade in the mid-1950s. A few years later the term "ribosome" was proposed by Richard B. Roberts. In 1974, Albert Claude, Christian de Duve and George Emil Palade were awarded the Nobel Prize for the discovery of the ribosome. Later, in 2009, Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath got the Nobel Prize in Chemistry for determining the detailed structure of ribosome. Since the 1960s, biochemical and mutational studies of the ribosome have made it possible to describe many of its functional and structural features.

Figure 1 .

 1 Figure 1. The small ribosomal subunit with tRNA binding sites (A, P and E) and decoding center.

Figure 2 .

 2 Figure2. The large subunit major functional sites are the tRNA binding sites (A, P and E), the peptide exit tunnel that extends through the body of the large subunit, and the peptidyl transferase center (PTC)[START_REF] Melnikov | One core, two shells: bacterial and eukaryotic ribosomes[END_REF].

Figure 4 .

 4 Figure 4. Ribosomal proteins of the 40S subunit that are colored according to conservation: protein cores found in all kingdoms are in light blue, proteins with archaeal homologs are in gold and unique for eukaryotes protein extensions are in red[START_REF] Rabl | Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1[END_REF].

Figure 5

 5 Figure5Ribosomal proteins of the 60S subunit that are colored according to conservation: protein cores found in all kingdoms are in light blue, proteins with archaeal homologs are in gold and unique for eukaryotes proteins extensions are in red. Zinc ions are shown as green spheres[START_REF] Klinge | Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6[END_REF].

Figure 6 .

 6 Figure 6. Sequence and secondary structure of rRNA of LSU from S. cerevisiae. Roman numerals denote different rRNA domains. Expansion segments are designated as the "ES", where the first digit is the location of the segment, and the last means its name (from Ribovision website).

Figure 7 .

 7 Figure 7. Sequence and secondary structure of 18S from S. cerevisiae. Expansion segments are designated as the "es", where the first digit is the location of the segment, and the last means its name (from Ribovision website).

  IF2 and IF3. Initiation is a very conserved step among bacteria. It involves the orderly binding of mRNA, IFs 3, 2 and 1 factor and the fMet-tRNA fMet . After the accommodation of the start codon, the IFs depart, followed by the formation of the ribosome (also called 70S) by the association of the large ribosomal subunit (50S) to the small (30S) (from review Rodnina, 2018, Fig.8).

Figure 8 .

 8 Figure 8. Scheme of the initiation process in bacteria. A detailed description is given in the text.

Figure 9 .

 9 Figure 9. Translation initiation process in eukaryotes. A detailed description is given in the text.

Figure 11 .

 11 Figure 11. This diagram shows the last 2 steps of translation termination and recycling for prokaryotes.

Figure 12 .

 12 Figure 12. This diagram shows termination and recycling processes for eukaryotes that are different from prokaryotes.

Figures

  Figures 13 and 14 summarize the translation process in both prokaryotes and eukaryotes.

Figure 13 .

 13 Figure 13. Schematic diagram of translation cycle in bacteria. The main steps of translation process are shown. Details regarding each step are provided in the main text.

Figure 14 .

 14 Figure 14. The translation cycle for eukaryotes which is divided into four main stages: initiation (including preinitiation), elongation, termination and recycling. Further details are also given in the text.

Figure 15 .

 15 Figure15. The process of the initiation complex formation based on FRET experiments(Milon et al., 2012).

Figure 17 .

 17 Figure17. The structure of IF1 from S. aureus determined with multidimensional NMR spectroscopy, where helices are colored in yellow, strands in green and coils in grey (2N8N fromKim et al., 2016).

Figure 18 .

 18 Figure 18. X-ray structure of translation IF2 from Methanothermobacter thermautotrophicus, where helices are colored in yellow, strands in green and coils in grey. Domains are indicated in the picture. More detailed information is provided in text (1G7R from Roll-Mecak et al., 2000).

Figure 19 .

 19 Figure 19. Cryo-EM structure of IF3 from Thermus thermophilus, where helices are colored in yellow, strands in green and coils in grey. Domains are indicated in the picture (5LMO from Hussain et al., 2016).

Figure 20 .

 20 Figure 20. Comparison of initiator (A) and elongator (B) methionine-tRNAs. The regions important for initiator tRNA are highlighted. Details are given in the text (Søgaard, 2005).

(

  Thermus thermophilus, Staphylococcus aureus and Escherichia coli) are similar. In initiation factor 1 the C-terminal region is very conserved, however, the N-terminal region in S. aureus is longer than in T. thermophilus and shorter than in E. coli. Already indicating speciesspecific structural differences (Fig 22).

Figure 22 .

 22 Figure 22. Scheme of the initiation factor 2 structure from T. thermophilus, S. aureus and E. coli.

Figure 23 .

 23 Figure 23. A scheme shows at which stage of protein synthesis different antibiotics bind to the ribosome (adapted from Wilson, 2014).

  The first-line treatment against methicillin-susceptible strains (MSSA) are β-lactamaseresistant penicillins (methicillin, oxacillin, cloxacillin, and flucloxacillin) or non-β-lactam antibiotics (clindamycin and co-trimoxazole) against methicillin-resistant strains (MRSA). To treat more serious invasive infections glycopeptide antibiotics are used (vancomycin and teicoplanin) that are administered intravenously. They are toxic and should be regularly monitored with a blood test. Some representative examples of antibiotics and their mechanism of resistance are shown in the Table4below.

Figure 24 .

 24 Figure 24. Sucrose gradient profile. The absorbance was measured by Nanodrop 2000c (Thermo Scientific). The black line indicates pooled 70S fractions.

Figure 25 .

 25 Figure 25. One-dimensional PAGE of S. aureus 70S ribosome.

Figure 28 .

 28 Figure 28. Sucrose gradient profile. The absorbance was measured by Nanodrop 2000c (Thermo Scientific). The black line indicates pooled 30S fractions.

Figure 29 .

 29 Figure 29. One-dimensional PAGE of S. aureus 30S.

Figure 30 .

 30 Figure 30. Agarose gel of 30S ribosome. The band correspond to 16S rRNA.

  transcriptase will be blocked by the ribosome, synthesized short cDNA fragments called toeprints. The mRNA that was used for this experiment was 225 nt in length, UUG start codon and open Shine-Dalgarno (SD) sequence. This mRNA has the ability to easily bind to the ribosome, due to the strong SD sequence.

Figure 31 .

 31 Figure 31. Formation of simplified translation initiation complex by toeprinting assays. Lanes U, C, G, A are sequencing ladders. The toeprint at position +16 is shown by an arrow. Experimental conditions were described in Fechter et al., 2009.

Figure 33 .

 33 Figure 33. Structure of spa mRNA.

Figure 34 .

 34 Figure 34. 6% PAGE of spa mRNA (225nts).

Figure 36 .

 36 Figure 36. One-dimensional PAGE of MTF (30 kDa).

Figure 38 .

 38 Figure 38. Schematic representation of basic composition of electron microscope.

Figure 39 .

 39 Figure 39. Representation of the cryo-EM grid at different scales (adapted from Wang and Sigworth, 2006).

Figure 40 .

 40 Figure 40. Scheme of sample analysis by cryo-electron microscopy.

  density map determined by cryo-EM. The most common metric for estimation global resolution is the Fourier shell correlation (FSC) that is calculating the resolution by comparing the structure form divided datasets with the obtained structure, but this approach cannot account for variance in the local resolution of difference areas of the cryo-EM map. For local resolution estimation ResMap, MonoRes or other tools are used. All these approaches require a considerable amount of processing time and in some cases their estimates are significantly different from one another.

Figure 41 .

 41 Figure 41. Principle steps of image processing in CryoSparc.

Figure 43 .

 43 Figure 43. The quantification of the toeprint (given in %) was first normalized according to the fulllength extension product bands using the ImageQuant TL software.

Figure 42 .

 42 Figure 42. Formation of simplified initiation complex with IF1 and IF2 by toeprinting assays. Lanes U, C, G, A are sequencing ladders. The toeprint at position +16 is shown by an arrow. A more detailed explanation is given in the text.

Figure 44 .

 44 Figure 44. Formation of simplified initiation complex with IF2 by toeprinting assays. Lanes U, C, G, A are sequencing ladders. The toeprint at position +16 is shown by an arrow.

Figure 45 .

 45 Figure 45. The quantification of the toeprint (given in %) was first normalized according to the full-length extension product bands using the ImageQuant TL software.

Figure 46 .

 46 Figure 46. Formation of initiation complex with fMet-tRNA fMet and Lys-tRNA by toeprinting assays. Lanes U, C, G, A are sequencing ladders. +16 and +19 positions are shown by arrows.

Figure 48 .Figure 47 .

 4847 Figure 48. Sucrose gradient profile (A). The black line on gradient indicates pooled 30S fractions. No 16S degradation was observed on agarose gel (B).

  seconds on Vitrobot apparatus (FEI, T = 4 o C, humidity 100%, blot force 5, blot waiting time 30 seconds) and vitrified in liquid ethane pre-cooled in liquid nitrogen. Data were collected on the Krios microscope (Titan, K2 Camera) and processed with Scipion package:

Figure 49 .

 49 Figure 49. Micrograph of SSU from S. aureus.

Figure 51 .

 51 Figure 51. Sucrose gradient profiles. 100 mM and 200 mM of NH4Cl and KCl and 400 mM of NaCl have been checked.

Figure 50 .

 50 Figure 50. 3D classification of 643532 particles divided into 10 classes. Particles were rejected because they were artifacts due to automatic picking during image processing.

Figure 52 .

 52 Figure 52. 15% polyacrylamide gel. The detailed information is in the text.

Figure 53 .

 53 Figure 53. Sucrose gradient profile (A) and agarose gel of 16S rRNA. No ribosomal RNA degradation was observed (B).

Figure 54 .

 54 Figure 54. 3D classification of 479872 particles divided into 10 classes. Obtained 1 class with 60000 particles was reclassified into 6 classes. The final class was obtained at 10 Å after refinement step. Particles were rejected because they were artifacts due to automatic picking during image processing.

Figure 56 .Figure 55 .

 5655 Figure 56. During 3D classification 1 class with 40000 particles was obtained and reclassified into 4 classes. Low resolution structures with IF3 in different conformations and IF2 were obtained. Particles were rejected because they were artifacts due to automatic picking during image processing.

Figure 57 .

 57 Figure 57. Sucrose gradient profiles. 30 mM and 50 mM NH4Cl have been checked.

Figure 58 .

 58 Figure 58. Scheme of stepwise assembling of initiation complex.

Figure 59 .

 59 Figure 59. Structures obtained after 4th data collection: 30S without spermidine.

Figure 60 .

 60 Figure 60. Structure obtained after 4th data collection: 30S with spermidine (35146 particles). The atomic model was taken from Khusainov et al., 2016 (5LI0, rRNA is in yellow color, r-proteins are in orange).

6Figure 61 .Figure 62 .

 6162 Figure 61. Structure obtained after 5th data collection (246049 particles): (A) cryo-EM reconstruction of 30S with mRNA without spermidine, (B)close-up view on the mRNA, (C) estimated resolution in CryoSparc software. rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red colors(Khusainov et al., 2016, 5LI0).

Figure 63 .Figure 64 .

 6364 Figure 63. Structure obtained after 6 th data acquisition: (A) 30S with mRNA, IF1 and IF3 complex reconstruction (15 min incubation), (B) close-up view on mRNA, (C) the estimated resolution in CryoSparc software.rRNA is highlighted in yellow, r-proteins are in orange, mRNA is in red and IF3 is in pink color (5LI0). IF1 was not detected.

Figure 69 .

 69 Figure69. Structure obtained after 6th data collection: 30S with mRNA, fMet-tRNA fMet and IF3 (A) with close-up view on mRNA (B). IF1 and IF2 were not detected (complex 5). rRNA is highlighted in yellow, rproteins are in orange, mRNA is in red, IF3 is in pink and fMet-tRNA fMet is in green colors (5LI0, 279232 particles). The resolution was estimated in CryoSparc software (C).

Figure 70 .

 70 Figure 70. Scheme of stepwise assembling of initiation complex

Figure 71 .

 71 Figure 71. Sucrose gradient profile. The control (without GMP-PNP and mRNA, with 5 mM Mg(OAc)2) is shown by the orange line (I gradient). The sample with 10 mM GMP-PNP and 5 mM Mg(OAc)2 is shown in green line (II gradient). The sample with previous conditions and additional mRNA is shown in grey line (III gradient). The sample with 10 mM GMP-PNP, additional mRNA and 10 mM Mg(OAc)2 is shown in blue line (IV gradient).

Figure 72 .

 72 Figure 72. 15% polyacrylamide gel: 30S colored in blue, 50S -in red, 70S -in green. Roman numerals indicate the gradient number from Figure58. On some lines, the sample is almost absent due to the low initial concentration. Some samples were concentrated with acetone that explains the waviness of some bands.

Figure 73 .

 73 Figure73. Agarose gel in native conditions of samples from III gradient (with 10 mM GMP-PNP, 5 mM Mg(OAc)2 and additional mRNA). First 2 lanes correspond to the 1 st peak (blue), third lane -to the second peak (red) and the last lane -to the third peak (blue). The upper and lower bands correspond to 50S and 30S subunits respectively.

Figure 74 .

 74 Figure 74. Structure obtained after 7th data collection. The ribosome was purified from cell extract with addition of GMP-PNP.

  Another interesting result is associated with 30S head conformation. It was observed that without adding spermidine during the ribosome or complex preparation the head conformation was open, while with spermidine it was closed allowing mRNA to properly accommodate inside the channel. This may also prove the need to add polyamines to the sample for the rRNA stabilization. The comparison of structures is shown in figures 76 and 77.

Figure 75 .

 75 Figure 75. Atomic model of E. coli ribosome (Noeske et al., 2015, 4YBB): rRNA is in light grey, 30S proteins are in blue, 50S proteins are in red. Helix 44 highlighted in yellow, putrescine compound is shown in sphere shape.

Figure 76 .

 76 Figure 76. Comparison of two 30S structures: without spermidine in yellow and with spermidine in light magenta colors.

Figure 77 .

 77 Figure 77. Comparison of two 30S with mRNA structures: without spermidine in yellow and with spermidine in light magenta colors. The mRNA region is stained darker than 30S.

Figure 78 .

 78 Figure 78. Comparison of two 30S with mRNA and IF3 structures: 15 minutes incubation of all components at once is in yellow and stepwise complex assembling in light magenta colors.

Figure 79 .

 79 Figure 79. Comparison of two 30S-mRNA structures: without IF3 is in yellow and with IF3 in light magenta colors.

Figure 80 .

 80 Figure 80. Sucrose gradient profile of C. albicans ribosome. The black line indicates pooled 80S fractions.

Figure 81 .

 81 Figure 81. A schematic phase diagram showing the solubility of a protein in solution as a function of the concentration of the precipitant present[START_REF] Asherie | Protein crystallization and phase diagrams[END_REF].

Figure 82 .

 82 Figure 82. Schematic representation of diffraction experiment (From www.imperial.ac.uk/x-raycrystallography).

Figure 86 .

 86 Figure 86. Crystals of 80S ribosome from C. albicans.

Figure 87 .

 87 Figure 87. Scheme of post-crystallization treatment.

Introduction

  La synthèse des protéines est un processus hautement conservé dont le ribosome est l'acteur principal. Pendant de nombreuses années, le ribosome est resté l'un des objets de recherche les plus importants pour les scientifiques du monde entier. Les structures à haute résolution des ribosomes et de ses sous-unités obtenues par cristallographie et microscopie électronique ont révolutionné le domaine de la biologie structurale. Le ribosome est le site de synthèse de protéines, également appelé traduction. Ce processus peut être divisé en 4 étapes principales: l'initiation, l'élongation, la terminaison et le recyclage. L'étape d'initiation de la traduction est une étape limitante, impliquant l'action de nombreux composants finement régulés. De nombreux antibiotiques agissent précisément à ce stade de la synthèse des protéines. L'étape d'élongation est mieux conservée par rapport à l'initiation chez toutes les espèces et inclut la formation de la chaîne polypeptidique. Les étapes de terminaison et de recyclage sont accompagnées de la libération de la chaîne polypeptidique et de la dissociation de tous les composants de la traduction.Projet de recherche 1La traduction chez les procaryotes a été principalement étudiée sur le modèle de bactéries gram-négatives telles qu'Escherichia coli (E. coli) et Thermus thermophilus. Cependant, des études récentes ont montré des différences significatives au niveau de l'étape d'initiation entre les bactéries gram négatives et les bactéries gram positives telles que Bacillus subtilis (B. subtilis) et Staphylococcus aureus (S. aureus). S. aureus est un agent pathogène humain fréquemment présent dans le nez, les voies respiratoires et la peau. Cette bactérie provoque des maladies telles que la pneumonie, la méningite, l'ostéomyélite et autres. Il a été signalé que 158 000 infections survenues en France en 2012 étaient dues à souche multirésistante Staphylococcus (Colomb-Cotinat et al., 2016). S. aureus présente une résistance extrêmement élevée aux antibiotiques ciblant les ribosomes.

1

  ) purification de tous les composants: 30S, IF (IF1, IF2 et IF3), ARNm et fMet-tRNA fMet , 2) détermination du meilleur rapport pour la formation de complexe, 3) acquisition de données, 4) résoudre la structure: traitement des données et construction du modèle atomique, la structure: traitement des données et construction du modèle atomique. Les protocoles pour les purifications des petites sous-unités ribosomales (30S) et des facteurs d'initiation (IF1, IF2 et IF3) ont été développés et validés. Les ribosomes se sont révélés purs, stables et capables de se cristalliser. Les facteurs d'initiation, l'ARNm et le fMet-tRNA fMet étaient suffisamment purs pour pouvoir être utilisés dans d'autres expériences. Les concentrations optimales de facteurs d'initiation du ribosome ont été trouvées et étaient de 3: 1 pour IF1 et IF2 et de 2: 1 pour IF3.Après plusieurs tentatives, la structure du 30S intact a été obtenue. L'ARNr a été stabilisé par addition de spermidine. Ensuite, l'ARNm a été ajouté au 30S et les structures ont été analysées en présence et en l'absence de spermidine. Le résultat obtenu montre le rôle de la spermidine dans la stabilisation de la conformation fermée 30S, aidant ainsi à loger correctement l'ARNm dans le canal. Plusieurs structures de 30S avec l'ARNm, le fMet-tRNA fMet et l'IF3 à différentes résolutions ont été obtenues. La structure de 30S avec l'ARNm, le fMet-tRNA fMet et l'IF2 à 10 Å a également été obtenue. Dans le même temps, IF1 n'a jamais été détecté.

  Détermination de la structure du ribosome 80S de Candida albicans par cristallographie aux rayons X Pour obtenir une structure ribosomique adaptée à l'étude d'antibiotiques spécifiques à l'espèce, plusieurs étapes doivent être complétées: 1) la croissance cellulaire et la purification des ribosomes doivent toujours être reproductibles, 2) une pureté d'échantillon élevée doit être obtenue, 3) les cristaux doivent avoir une taille suffisante pour le traitement post-cristallisation, 4) les cristaux avec une bonne diffraction doivent être analysés sur synchrotron. Les protocoles de purification et de cristallisation du ribosome pour C. albicans ont été adaptés à partir des protocoles pour S. cerevisiae (Ben Shem et al., 2011). La purification efficace du ribosome a été réalisée en plusieurs étapes: lyse cellulaire, précipitation du ribosome au polyéthylène glycol (PEG), séparation des 80S de ses sous-unités par des gradients de saccharose et dernière précipitation du PEG pour la concentration de l'échantillon. Les ribosomes obtenus se sont révélés purs, stables et susceptibles d'être cristallisés. Le criblage à grande échelle des conditions de cristallisation a été effectué. Le changement de la température de cristallisation de + 4 ° C à + 20 ° C a affecté négativement l'apparition des cristaux. Les changements significatifs affectant la forme et la taille des cristaux étaient les concentrations de ribosome, ainsi que la concentration en sels, le rapport entre l'échantillon et le réservoir à l'intérieur de la goutte et le volume du réservoir. Une

Table 1 .

 1 Composition and characteristics of ribosome from different species 13

Table 2 .

 2 List of ribosomal proteins from Candida albicans 15

Table 3 .

 3 Mechanisms of action and resistance mechanisms of selected ribosome-targeting antibiotics 38

Table 4 .

 4

Table 11 .

 11 Summary of tried crystallization conditions ABBREVIATIONS

	Methods :
	NMR	nuclear magnetic resonance
	Cryo-EM	cryo-electron microscopy
	FRET	Förster resonance energy transfer
	LC-MS/MS	liquid chromatography coupled mass spectrometry
	PAGE	polyacrylamide gel electrophoresis
	S. aureus :
	S. aureus	Staphylococcus aureus
	E. coli	Escherichia coli
	B. subtilis	Bacillus subtilis
	T. thermophilus	Thermus thermophilus
	B. stearothermophilus	Bacillus stearothermophilus
	MRSA	methicillin-resistant S. aureus
	MSSA	methicillin-susceptible S. aureus
	TSS	toxic shock syndrome
	Ribosome :
	aa-tRNA	aminoacyl-tRNA
	A-site	aminoacyl site
	EF	elongation factor (eIF -eukaryotic IF)
	E-site	exit site
	IF	initiation factor
	mRNA	messenger ribonucleic acid
	PTC	peptidyl transferase center
	P-site	peptidyl site
	RF	release factor
	RRF	ribosome recycling factor
	rRNA	ribosomal ribonucleic acid
	SD	Shine-Dalgarno
	tRNA	transfer ribonucleic acid
	Cryo-electron microscopy:
	CTF	contrast transfer function
	DDD	direct detector device
	FEG	field emission gun
	FSC	Fourier shell correlation
	TEM	transmission electron microscopy
	Reagents:
	DTT	dithiothreitol
	EDTA	ethylenediaminetetraacetic acid
	GDP	guanosine diphosphate
	GTP	guanosine triphosphate
	LB	lysogeny broth
	PEG	polyethylene glycol
	SDS	sodium dodecyl sulfate

Table 2 .

 2 List of ribosomal proteins from Candida albicans (www.uniprot.org), where e -eukaryotic-specific ribosomal proteins, u -universal proteins, MW -molecular weight, pI -isoelectric point.

		Small ribosomal subunit			Large ribosomal subunit
	Name of protein Name of gene	MW	pI	Name of protein Name of gene	MW	pI
	eS1	RPS1	29,0	10,04	uL1	RPL1	24,4	9,76
	uS2	RPS0	28,7	4,91	uL2	RPL2	27,3	10,71
	uS3	RPS3	27,3	9,23	uL3	RPL3	43,9	10,26
	uS4	RPS9	21,7	10,20	uL4	RPL4	39,2	10,74
	eS4	RPS4	29,4	10,15	uL5	RPL11	19,8	9,95
	uS5	RPS2	26,9	10,24	uL6	RPL9	21,7	9,51
	eS6	RPS6	27,1	10,15	eL6	RPL6	19,8	10,21
	eS7	RPS7	21,2	9,92	eL8	RPL8	28,5	10,05
	uS7	RPS5	25,2	8,7	uL10	RPP0	33,3	4,83
	eS8	RPS8	22,7	11,09	uL11	RPL12	17,8	9,51
	uS8	RPS22	14,8	9,88	uL13	RPL16	22,6	10,32
	uS9	RPS16	15,7	10,29	eL13	RPL13	23,0	10,61
	eS10	RPS10	13,8	9,83	eL14	RPL14	14,7	10,90

Table 3 .

 3 Mechanisms of action and resistance mechanisms of selected ribosome-targeting antibiotics. DMdrug modification/ degradation, E efflux/membrane permeability, EF elongation factor, FP factor-assisted protection, ND not determined, PTC peptidyl-transferase center, TA target alteration via modification, TM target mutation (adapted from[START_REF] Wilson | Ribosome-targeting antibiotics and mechanisms of bacterial resistance[END_REF].

	Antibiotic	Molecular	Inhibition mechanism	Resistance
		target		mechanisms
	Chloramphenicol	50S	PTC	DM, E, TA, TM
	Clindamycin,	50S	PTC	DM, E, TA, TM
	lincomycin
	Dalfopristin,	50S	PTC	E, TA, TM
	quinupristin
	Doxycycline, tigecycline	30S	tRNA delivery	DM, E, Fp, TM
	Edeine	30S	Initiation	DM, TM
	Evernimicin, avilamycin	50S	Initiation	TA, TM
	Kanamycin	30S	Initiation	DM
	Kasugamycin	30S	Initiation	DM, TA, TM
	Linezlid	50S	PTC	E, TA, TM
	Puromycin	50S	PTC	DM
	Sparsomycin	50S	PTC	E, TM
	Spectinomycin	30S	Translocation	DM, E, TA, TM
	Streptomycin	30S	Translocation	DM, E, TA, TM
	Thermorubin	70S	Initiation	ND
	Thiostrepron	50S	Factor binding	TA, TM
	Tiamulin	50S	PTC	E, TA, TM
	Tobramycin	30S	Initiation	DM, E
	Viomycin, capreomycin	70S	Translocation	DM, TA, TM

Table 4 .

 4 Antibiotics used against S. aureus and the mechanism of their resistance[START_REF] Rayner | Antibiotics currently used in the treatment of infections caused by Staphylococcus aureus[END_REF][START_REF] Johnson | Antimicrobial agents in treatment of MRSA infections[END_REF][START_REF] Morgan | Treatment of MRSA soft tissue infections: an overview[END_REF].

	Name	Mechanism of resistance
	Penicillin	β-lactamase, enzymatic inactivation of penicillin
	Methicillin	Expression of new penicillin-binding protein
	Tetracycline	Efflux from cell, modification of ribosome
	Chloramphenicol	Enzymatic inactivation
	Erythromycin	Enzymatic modification of ribosomal RNA. Prevents drug
		binding to ribosome
	Streptomycin	Mutation in ribosomal protein, prevents drug binding, enzymatic
		inactivation
	Kanamycin, Gentamicin	Enzymatic inactivation
	Trimethoprim	Alternative dihydrofolate reductase
	Mupirocin	Alternative isoleucyl-tRNA
	Fluoroquinolones	Altered DNA gyrase, efflux
	Antiseptics	Efflux

Table 5 .

 5 List of ribosomal proteins found in S. aureus ribosome sample by LC-MS/MS analysis, where bbacterial specific proteins, u -universal proteins.Figure 26. Agarose gels in native (A) and denaturing conditions (B). On native gel the upper and lower bands correspond to 50S and 30S. On denaturing gel of 70S (1 and 2 μg) the upper and lower bands correspond to 23S

		Small ribosomal subunit proteins		Large ribosomal subunit proteins
	Protein	Spectral count	MW (kDa)	pI	Protein	Spectral count MW (kDa)	pI
	bS1	0	43,3	4,60	uL1	236	24,7	9,5
	uS2	260	29,1	5,3	uL2	380	30,1	11,3

Table 6 .

 6 List of non-ribosomal proteins found in S. aureus ribosome sample by LS-MS/MS analysis.

	Protein

Table 7 .

 7 Characteristics of microscopes used for data acquisition.

		Titan Krios	Tecnai F30 Polara	Talos Arctica
	Voltage (kV)	300	300	200
	Defocus range (µm)

Table 8 .

 8 List of proteins found in S. aureus 30S ribosome sample by LC-MS/MS analysis, where b -bacterial specific proteins, u -universal proteins.

	Protein	Spectral count	MW (kDa)	pI
	bS1	4	43,3	4,60
	uS2	258	29,1	5,3
	uS3	280	24,1	10,2
	uS4	328	23,0	10,4
	uS5	184	17,7	10,3
	bS6	139	11,6	4,9
	uS7	243	17,8	10,4
	uS8	120	14,8	9,8
	uS9	102	14,8	11,0
	uS10	128	11,6	10,2
	uS11	105	13,9	11,9
	uS12	194	15,3	11,9
	uS13	278	13,7	10,8
	uS14	71	7,3	11,6
	uS15	112	10,6	10,9
	bS16	75	10,2	10,4
	uS17	150	10,2	10,2
	bS18	96	9,3	11,7
	uS19	298	10,6	10,4
	bS20	266	9,0	11,0

Table 10 .

 10 List of proteins found in C. albicans 80S ribosome sample by LC-MS/MS analysis, where beukaryotic specific proteins, u -universal proteins.

	Small ribosomal subunit		Large ribosomal subunit
	Protein	Score	MW	pI	Protein	Score	MW	pI
	eS1_RPS1	240	29,0	10,04	uL1_RPL1		24,4	9,76
	uS2-RPS0	180,5	28,7	4,91	uL2_RPL2		27,3	10,71
	uS3_RPS3	331	27,3	9,23	uL3_RPL3		43,9	10,26
	uS4_RPS9	148	21,7	10,20	uL4_RPL4		39,2	10,74
	eS4_RPS4	190	29,4	10,15	uL5_RPL11		19,8	9,95
	uS5_RPS2	156	26,9	10,24	uL6_RPL9		21,7	9,51
	eS6_RPS6	233	27,1	10,15	eL6_RPL6		19,8	10,21
	eS7_RPS7	283	21,2	9,92	eL8_RPL8		28,5	10,05
	uS7_RPS5	285	25,2	8,7	uL10_RPP0		33,3	4,83
	eS8_RPS8	332	22,7	11,09	uL11_RPL12		17,8	9,51
	uS8_RPS22	201,5	14,8	9,88	uL13-RPL16	74	22,6	10,32
	uS9_RPS16	137	15,7	10,29	eL13-RPL13		23,0	10,61
	eS10_RPS10	94	13,8	9,83	eL14_RPL14		14,7	10,90

Table 11 .

 11 Summary of tried crystallization conditions.

	Component	Concentration		Types
	Buffers, pH 6.5 -7.5	0 -100 mM	Tris	Bis-tris
	Cations	0 -600 mM	K +	NH4 +
	Anions	0 -600 mM	SCN -	Acetate -

ACKNOWLEDGEMENTS

This thesis describe the work that I have been doing since 2015 under the supervision of several people.

First of all, I would like to thank my jury members Pr. Yves Mechulam, Dr. Fabien Darfeuille,

Crystallization

The purity of the sample was also checked by crystallization. The protocol was taken from Marat Yusupov's laboratory.

As a result, around 200 µm crystals were obtained which is an indicator of the purity of the sample (Fig. 27).

Cell growth

The vector pnEAvH containing infB gene from S. aureus was transformed into E. coli BL21(DE3). Cells were grown in LB medium with 100 µg/ml of ampicillin. The expression of IF2 was induced with 1 mM IPTG. Cells from 3 L culture were centrifuged and the pellet

One-dimensional PAGE

The obtained gels demonstrated high purity of the sample (Fig. 32).

Dialysis

The sample was dialyzed into buffer Q in order to remove imidazole from the sample.

Storage

The sample was kept at -20 o C in 50% glycerol.

Purified MetRS was checked on 15% PAGE (Fig. 35).

PERSPECTIVES

The work described in this manuscript is only the first step in understanding how the initiation process in Staphylococcus aureus occurs.

Two pathways of stepwise assembling of initiation complex described in the literature may be related to species-specific features [START_REF] Hussain | Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation[END_REF], Milon et al., 2012). It can be assumed that in some bacterial species (E. coli) with long N-domain of IF2, the binding of IF1 or IF2 depends on their mutual presence, while in other species (Th. thermophilus) initiation factors can bind independently. This can lead to different species-specific pathways of protein binding to the ribosome. Further research is required in order to confirm or refute this hypothesis.

It is absolutely imperative to complete ex vivo experiments that could clarify the mechanism of the initiation process in S. aureus. Also, using this method, it is possible that additional factor/component could be found for the proper initiation complex formation.

Another interesting feature of S. aureus is the presence of virulent RNAs, which can affect the initiation stage. The study of this process can have not only theoretical, but also practical significance, because virulent RNAs participate in the biofilm's formation, which complicates the treatment of diseases caused by this bacterium.

All the results obtained in this work will help to describe the initiation process in S. aureus and the gained knowledge will be used to develop compounds against pathogenic bacterium.

CONCLUDING REMARKS

Nowadays, studies of translation process in pathogenic bacteria have not only theoretical, but also practical significance. One example of pathogenic bacterium is Staphylococcus aureus that is responsible for severe nosocomial and community-acquired infections. The diseases caused by this bacterium include pneumonia, meningitis, endocarditis, osteomyelitis, septic arthritis and infections of bones and organs. Certain strains of S. aureus produce the superantigen TSST-1, which is responsible for the toxic shock syndrome (TSS). As a result, mortality rates are very high; death can occur within two hours. [START_REF] Chen | A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 chinese isolates[END_REF]. Also, this bacterium is highly resistant to most commonly used antibiotics. In 2014, a report came out from the World Health Organization that the antibiotic resistance is no longer a prediction for the future, the disease may appear now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance is now a major threat to public health.

At the moment, study of protein synthesis from human pathogen S. aureus is a hot topic.

The first ribosome structure from S. aureus was published only three years ago [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF]. Moreover, not only the ribosome is an interesting object to study. The initiation process as a rate limiting stage is also necessary to study. Despite all the difficulties that I have encountered, significant progress in the investigation of the initiation process was achieved.

The work described in this manuscript is only the initial step of studying the initiation process in S. aureus. The knowledge gained during my work will be used for further research.

The obtained results may also be used in the future to fight against this bacterium.

RESEARCH PROJECT 2

Structure determination of Candida albicans ribosome

RESULTS

One-dimensional PAGE

Gel demonstrates the absence of high molecular weight non-ribosomal proteins (Fig. 83).

Agarose gel in denaturing conditions

Agarose gel demonstrates the absence of degradation of rRNA from C. albicans (Fig. 84).

Mass spectrometry

The results from mass spectrometry analysis of 80S from C. albicans are summarized below (Table 10).