Md Shahidul Islam

Mark J Pallen

Stephen J W Busby

A cryptic promoter in the LEE1 regulatory region of enterohaemorrhagic Escherichia coli: promoter specificity in AT-rich gene regulatory regions

Keywords: bacterial promoters, transcription initiation, promoter discrimination, mutational analysis, bacterial multi-subunit RNA polymerase

Transcription of the LEE1 operon in the locus of enterocyte effacement of enterohaemorrhagic Escherichia coli is due to the P1 promoter. Mutational and biochemical analyses reveal the existence of an overlapping promoter, designated P1A, which can drive transcript initiation 10 base pairs upstream from the P1 promoter transcript start point. Because of the overlap between P1 and P1A, P1A activity is unmasked only when the P1 promoter is inactivated by mutation. We report here that mutation of the P1 -10 element is less effective in unmasking P1A promoter activity than mutation of the P1 -35 element. This suggests that the P1 promoter -35 element, which corresponds to the consensus, can sequester RNA polymerase even when P1 is inactive and, thereby, prevent RNA polymerase from serving the P1A promoter. We propose that such promoter elements may play a role in enforcing specificity in bacterial regulatory regions that contain alternative possible promoters.

INTRODUCTION

Bacterial gene regulatory regions contain promoters that have evolved to set the level of expression of downstream genes, and the different promoter elements that are recognised by the multi-subunit bacterial RNA polymerase are now well understood. Exhaustive analyses of Escherichia coli promoters have shown that the principal promoter elements are the -35 and -10 hexamer elements, and consensus sequences of TTGACA and TATAAT respectively have been derived. Both elements are recognised directly by determinants in the RNA polymerase ı subunit and mutations in either element can lead to big reductions in promoter activity. The A at position 2 of the -10 element is especially important as it is implicated in opening of the transcription 'bubble' as well as promoter recognition. Thus, to a first approximation, the strength of any E. coli promoter will depend on the degree to which the base sequences of the functional -10 and -35 elements correspond to the consensus (reviewed in [START_REF] Busby | Where it all begins: an overview of promoter recognition and open complex formation[END_REF]).

Computational analysis of E. coli sequences has revealed the existence of potential -10 and -35 elements at higher than expected frequencies in intergenic regulatory regions [START_REF] Huerta | Sigma70 promoters in Escherichia coli: specific transcription in dense regions of overlapping promoter-like signals[END_REF]. Whilst this is probably a by-product of the evolution of gene regulatory regions, it raises the issue of the specificity of promoter recognition by RNA polymerase, and the role played by cryptic promoter elements. This is especially relevant for regulatory regions within horizontally-acquired pathogenicity islands that are generally characterised by a high proportion of AT base pairs [START_REF] Hacker | Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution[END_REF] and hence contain many potential -10 elements.

In this work, we have focussed on a short promoter fragment, taken from an AT-rich pathogenicity island, the locus of enterocyte effacement (LEE) of enterohaemorrhagic E. coli (EHEC) serotype O157:H7. Recall that EHEC serotype O157:H7 causes haemorrhagic colitis and haemolytic uremic syndrome in humans, and the LEE is one of its key pathogenicity determinants [START_REF] Elliott | The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69[END_REF]. This promoter fragment carries the principal promoter, P1, responsible for the expression of one of the operons encoded by the LEE, LEE1. In a recent study, we used mutational analysis to identify the LEE1 P1 promoter -10 and -35 hexamer elements [START_REF] Islam | Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA[END_REF]. Here we report the existence of a second promoter that overlaps LEE1 P1, whose activity is unmasked when the LEE1 P1 promoter is inactivated by mutation. Surprisingly, we found that mutations in the LEE1 P1 promoter -10 and -35 hexamer elements unmask this promoter to different extents. Hence, even when the LEE1 P1 promoter is inactive due to a -10 element mutation, the activity of the alternative promoter is very low due to sequestration of RNA polymerase by the LEE1 P1 promoter -35 element.

EXPERIMENTAL

Bacterial strains, plasmids, promoter fragments and primers E. coli K-12 strain M182, which carries a deletion of the entire lactose operon [START_REF] Casadaban | Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu[END_REF], was used throughout this work, and was grown on MacConkey lactose indicator plates. The LEE20-275 EcoRI-HindIII fragment, which was constructed in our previous work [START_REF] Islam | Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA[END_REF], contains 120 base pairs of the EHEC O157:H7 LEE1 operon regulatory region, including the LEE1 P1 promoter, and is illustrated in Figure 1A. The vector plasmids used for cloning this fragment and derivatives with different mutations were pRW224, a lac expression vector encoding resistance to tetracycline [START_REF] Islam | Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA[END_REF][START_REF] Lodge | Broad host range plasmids carrying the Escherichia coli lactose and galactose operons[END_REF], and pSR, a colE1-based ϯ general cloning vector, encoding resistance to ampicillin [START_REF] Kolb | Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation[END_REF]. Promoters that are cloned into pSR on EcoRI-HindIII fragments run into the bacteriophage Ȝ oop terminator downstream of the HindIII site [START_REF] Browning | Assays for transcription factor activity[END_REF]. Mutations were introduced into the LEE20-275 fragments cloned in pRW224 by using error prone PCR [START_REF] Leung | A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction[END_REF] with flanking primers D10520 (5'-CCCTGCGGTGCCCCTCAAC-3') and D53463 (5'-GGGGGATGTGCTGCAAGGCG -3'), or by using megaprimer PCR [START_REF] Sarkar | The "megaprimer" method of site directed mutagenesis[END_REF] as described by Chismon et al. [START_REF] Chismon | Unusual organisation, complexity and redundancy at the Escherichia coli hcp-hcr operon promoter[END_REF]. The different bases at the LEE1 P1 promoter in the LEE20-275 fragment are numbered 1-120, and mutations are denoted by their position and the substituted base, as shown in Figure 1.

Promoter activity assays

E. coli M182 containing pRW224 carrying different mutated LEE20-275 fragments was inoculated from single colonies on MacConkey lactose indicator plates into LB medium supplemented with 35 μg ml -1 tetracycline. Cultures were grown aerobically with shaking at 37°C, harvested during exponential growth, and β-galactosidase expression was measured by the Miller method [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. Recorded activities are the average of at least three independent experiments and are taken as a measure of the promoter activity of the cloned LEE20-275 fragment.

In vitro experiments

The in vitro transcription experiments were performed as described by Browning et al. [START_REF] Browning | Assays for transcription factor activity[END_REF] using PstI-BamHI DNA fragments purified from caesium chloride preparations of pSR vector plasmid carrying the starting or mutated LEE20-275 fragment. These fragments served as a template for multiple round in vitro transcription assays in which 20 ng fragment was incubated in transcription buffer containing 40 mM Tris pH 7.9, 10 mM MgCl 2 , 1 mM DTT, 100 mM KCl, 100 µg ml -1 bovine serum albumin, 200 µM GTP, 200 µM ATP, 200 µM CTP, 10 µM UTP and 5 µCi [α 32 P]-UTP. Reactions were started by adding holo E. coli RNA polymerase, purchased from Epicenter (Madison). RNA products were analysed on a denaturing 5.5% polyacrylamide gel and visualised using a Fuji phosphor screen and BioRad Molecular Imager FX. Gels were calibrated with the pSR plasmid-encoded 108 base RNA-I transcript and Maxam-Gilbert "G+A" sequencing reactions. Permanganate footprints were performed using the PstI-BamHI DNA fragments labelled at the BamHI end using [Ȗ 32 P]-ATP and polynucleotide kinase. Experiments were performed following the protocol of Savery et al. [14] using holo E. coli RNA polymerase from Epicenter (Madison). Each reaction contained approximately 3 nM labelled DNA fragment in 20 mM HEPES, pH 8.0, 5 mM MgCl 2 , 50 mM potassium glutamate, 1 mM DTT and 0.5 mg ml -1 BSA and 50 nM holo RNA polymerase as required. Products of footprinting reactions were analysed on denaturing 6% polyacrylamide sequencing gels, calibrated with Maxam-Gilbert "G+A" sequencing reactions.

RESULTS AND DISCUSSION

Unexpected promoter activity due to the 64G and 98C substitutions in the LEE20-275 fragment

The LEE20-275 EcoRI-HindIII DNA fragment, illustrated in Figure 1A, covers the LEE1 P1 promoter. The promoter activity of this fragment can be measured by using the pRW224 lac expression vector plasmid, and pRW224 carrying the LEE20-275 fragment confers a Lac + phenotype on the E. coli K-12 Δlac strain, M182. In recent work [START_REF] Islam | Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA[END_REF], error-prone PCR was exploited to generate a library of random mutations in this fragment cloned in pRW224. We then used MacConkey indicator plates to screen the Lac phenotype of colonies of M182 cells carrying the pRW224 library and selected for single base substitutions that reduced promoter activity and gave Lac -colonies. Strikingly, over 90% of the selected base changes fell in two hexamer elements, TTGACA and TACACA, that were identified as the P1 promoter -35 and -10 hexamers respectively (Figure 1A). Assays with the different mutated LEE20-275 fragments cloned in pRW224 showed that single mutations in the P1 -10 element reduced measured lac expression to 3-6% of the level with the starting fragment, whilst mutations in the -35 element reduced expression to 12-27% of the starting level [START_REF] Islam | Organization of the LEE1 operon regulatory region of enterohaemorrhagic Escherichia coli O157:H7 and activation by GrlA[END_REF].

In complementary experiments, site-directed mutagenesis was used to construct specific mutations in the LEE20-275 fragment, including the 98C mutation at position 2 of the P1 -10 hexamer. Whilst screening Lac -M182 colonies that carried pRW224 with the LEE20-275 98C mutant fragment, we unexpectedly found a single Lac + colony. Sequence analysis showed that, in this colony, the LEE20-275 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. ϰ fragment contained a second accidentally generated substitution, 64G, located 34 base pairs upstream of the 98C base change (Figure 1B). Table 1 lists measurements of the promoter activity of this fragment, together with controls. As expected, the 98C substitution reduces expression to 4% of the starting level, whilst the measured promoter activity of the LEE20-275 fragment with both the 98C and 64G substitutions is 20-fold higher, which is nearly 80% of the starting level. Data in Table 1 also show that, in isolation, the 64G mutation causes but a modest 14% increase in promoter activity.

Characterisation of the LEE1 P1A promoter

Since it is known that many E. coli promoters are completely inactivated by base changes at position 2 of the -10 hexamer [START_REF] Rosenberg | Regulatory sequences involved in the promotion and termination of RNA transcription[END_REF][START_REF] Miroslavova | Investigations of the modular structure of bacterial promoters[END_REF][START_REF] Jayaraman | Mutational analysis of nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli[END_REF], the simplest explanation for the effect of the 64G mutation is that it unmasks a cryptic promoter in the LEE20-275 fragment. To investigate this, transcripts formed, after labelled nucleoside triphosphates were added to binary complexes of purified RNA polymerase and the LEE20-275 fragment, were analysed. In this experiment, RNA polymerase runs to a downstream transcription terminator, and the RNA transcripts are labelled by using [α 32 P]-labelled UTP and analysed by gel electrophoresis (Figure 2A). With the starting LEE20-275 fragment, the major transcript is ~102 bases, which corresponds to the LEE1 P1 transcript that starts at position 107A (Figure 1A). As expected, this transcript is completely absent when the DNA fragment carries the 98C substitution. However, with the LEE20-275 fragment carrying both the 98C and 64G substitutions, a new ~112 base transcript is observed. This corresponds to a transcript starting at position 97T, suggesting that the 64G substitution has unmasked a promoter (Figure 1B). To confirm this, we used potassium permanganate footprinting to compare regions of DNA duplex unwinding in binary complexes of purified RNA polymerase and the LEE20-275 fragment either without or with the 98C and 64G substitutions. Recall that potassium permanganate modifies T residues in the single stranded 'bubble' produced after local unwinding of promoter DNA around the transcription start at promoters [14]. Results in Figure 2B show that, without the substitutions, there is extensive unwinding that starts just downstream of the P1 promoter -10 hexamer, and that this unwinding is suppressed by the 98C substitution. In contrast, with the fragment carrying both the 98C and 64G substitutions, clear duplex opening is seen at positions 86, 88, 89 and 93, just upstream from the transcript start at 97T. This argues that 98C and 64G substitutions cause RNA polymerase to recognise a new promoter (P1A), with a transcript start that is 10 base pairs upstream from the P1 promoter start (Figure 1B).

In order to identify sequence elements essential for the P1A promoter, error prone PCR was used to generate 3 independent preparations of the LEE20-275 fragment with the 98C and 64G substitutions. They were cloned into pRW224, the mixture of resulting recombinant plasmids was transformed into E. coli strain M182, and transformants were grown on MacConkey indicator plates. As expected, the majority of colonies scored as Lac + , but, after screening over 15,000 transformants, we identified 4 Lac - colonies that each carried a supplementary base change. These were an A for C change at position 88 (88C), and A for G substitution at position 89 (89G), a T for C substitution at position 90 (90C), and the insertion of a single T between positions 71 and 72 (Figure 1B). Table 2 shows measurements of the promoter activity of each of the four mutant fragments. The data show that the 88C substitution reduces promoter activity by over 20-fold, whilst the other three substitutions have smaller effects. These data, together with the in vitro results in Figure 2, identify the hexamer, TAATGT, from position 87 to position 92 as the P1A -10 hexamer, upstream of the transcript start at position 97. Hence, the 88C mutation at position 2 of the hexamer has the biggest effects on P1A activity and is within the region of duplex unwinding following RNA. We tentatively assign TGGTTT from position 62 to position 67 as the P1A -35 hexamer (Figure 1B). Although this corresponds to the consensus TTGACA at only 2 out of 6 positions, the concordant G at position 64 is the G that was created by the 64G substitution that unmasked the P1A promoter. Thus, the high promoter activity of the LEE20-275 fragment carrying the 98C and 64G substitutions is likely due to improvement of the -35 element from TGATTT to TGGTTT.

Activity of LEE1 P1A without the 64G substitution

The chance isolation of the 64G substitution in the LEE20-275 fragment, in combination with the 98C substitution, led us to identify the P1A promoter. We wanted to understand whether P1A had been generated by the 64G mutation, or whether it is a cryptic promoter, present in the starting LEE20-275 fragment, but silenced because of competition by the P1 promoter. To investigate this, we exploited the ϱ 88C substitution at position 2 of the P1A promoter -10 hexamer element that inactivates P1A. Table 3 illustrates an experiment designed to quantify the activity of P1A in the LEE20-275 fragment and in different mutated derivatives, by measuring the effect of the 88C substitution on β-galactosidase expression after the fragments were cloned into pRW224.

Results in Table 3 show that the 88C substitution has very little effect on the measured promoter activity of the starting LEE20-275 fragment. However, when the LEE20-275 fragment carries mutations in the P1 -10 element (98C), or in the P1 -35 element (76G), or in both elements of P1 (76G 98G), the 88C substitution reduces expression by over 85%. The simplest explanation for these observations is that P1A is a cryptic promoter that is silenced by competition from P1, but becomes active as P1 is inactivated.

Surprisingly, the introduction of the 76G substitution in the P1 -35 element substantially increased the activity of the LEE20-275 fragment carrying the 98G substitution in the P1 -10 element. This increased expression is completely suppressed by the 88C substitution, and, hence, must be due to the P1A promoter. From this, we conclude that, even though the P1 promoter is inactivated by the 98G substitution, the activity of P1A remains low because RNA polymerase still makes abortive contacts with the P1 consensus TTGACA -35 hexamer element. Hence, P1A activity increases as these interactions are weakened, due to substitutions such as 76G. In a related experiment, one of the 7 consecutive T residues immediately upstream of the P1 promoter -35 hexamer element (Δ71T) was deleted, in order to change the length of the spacer between the P1A promoter -10 and -35 hexamer elements closer to the optimal 17 base pairs [START_REF] Rosenberg | Regulatory sequences involved in the promotion and termination of RNA transcription[END_REF]. Results in Table 3 show that the promoter activity of the resulting LEE20-275 fragment carrying the Δ71T change is reduced ~3-fold by the 88C substitution. Hence strengthening P1A in the LEE20-275 fragment increases its contribution to measured promoter activity.

Conclusions

Many bacterial gene regulatory regions, especially those that are AT-rich, contain multiple promoters, and it is generally assumed that they are a by-product of evolution. Here we have identified a cryptic promoter, P1A, which overlaps the LEE1 P1 promoter of EHEC serotype O157:H7. Our results suggest that the P1A -10 hexamer element lies between the P1 -10 and -35 hexamer elements, and the P1A -35 hexamer element lies upstream of the P1 -35 element (Figure 1). The P1A promoter was discovered following the chance isolation of the 64G substitution that improves its -35 element. Our data argue that the P1A promoter can function even without the 64G substitution and thus it is a true cryptic promoter. The P1A promoter overlaps with P1 and hence the two promoters compete and are mutually exclusive. In our conditions, with the starting LEE20-275 fragment, the P1 promoter clearly wins the competition. However, as P1 is weakened, or P1A is strengthened, the scale tips towards P1A. Interestingly, when P1 is inactivated by a substitution at position 2 of its -10 hexamer, measured P1A activity remains low due to the P1 -35 element, which corresponds exactly to the consensus. Thus the P1 promoter, even when inactive, can retain the ability to sequester RNA polymerase, thereby blocking access to the P1A promoter.

Potentially, clusters of potential promoter elements in regulatory regions can have many different consequences. For example, they may, quite simply, give rise to multiple transcription starts and recent high resolution analysis of the E. coli transcriptome has identified many cases of this [START_REF] Mendoza-Vargas | Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli[END_REF]. This can lead to complex patterns of regulation. Alternatively, these elements could be the remains of now defunct regulatory systems, or be awaiting future adoption. Our finding that the P1 -35 element retains function even when P1 is inactive, together with similar observations with the E. coli gal operon regulatory region [START_REF] Johnston | Binding of Escherichia coli RNA polymerase to a promoter carrying mutations that stop transcription initiation[END_REF] underscores that residual promoter elements at non-functional promoters can affect the distribution of RNA polymerase. At present, the role of the LEE1 P1A is unclear. However, we note that, although consensus -35 hexamer elements are rare at E. coli promoters [START_REF] Mitchell | Identification and analysis of 'extended -10' promoters in Escherichia coli[END_REF], the LEE1 P1 -35 element is conserved in all of the pathogenic E. coli listed in the xBASE database that contain a locus of enterocyte effacement [START_REF] Chaudhuri | xBASE2: a comprehensive resource for comparative bacterial genomics[END_REF], as well as in Citrobacter rodentium [START_REF] Schauer | Attaching and effacing locus of Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia[END_REF]. We speculate that the rationale for retaining TTGACA as the LEE1 P1 -35 element is to focus RNA polymerase at P1 and to reduce the use of neighbouring promoters such as P1A. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1. Mutational analysis of the LEE20-275 promoter fragment

The table shows measured ȕ-galactosidase activities in cultures of E. coli strain M182 carrying pRW224 containing the LEE20-275 (WT) fragment and different derivatives. Cultures were grown aerobically at 37°C in LB medium to an optical density of ~0.5 at 65 0 nm. Activities were measured in triplicate, giving a mean and standard deviation (SD). Activities expressed as a percentage of activity with the starting LEE20-275 fragment are shown in parentheses. The central part of the table shows the fragment base sequence from position 62 to position 102, with the P1 promoter -10 and -35 hexamer elements grey shaded. Base substitutions in the different fragments are underlined and highlighted in boldface type.

. Identification of the functional elements of the P1A promoter

The table shows measured ȕ-galactosidase activities in cultures of E. coli strain M182 carrying pRW224 containing the LEE20-275 64G 98C fragment and different mutations. Cultures were grown aerobically at 37°C in LB medium to an optical density of ~0.5 at 65 0 nm. Activities were measured in triplicate, giving a mean and standard deviation (SD). Activities expressed as a percentage of activity with the starting LEE20-275 64G 98C fragment are shown in parentheses. The central part of the table shows the fragment base sequence from position 62 to position 102, with the location of P1 promoter -10 and -35 hexamer elements grey shaded and predicted cryptic promoter -10 and -35 elements are underlined.

Base substitutions and insertion in the different fragments are highlighted in boldface type. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 08 Apr 2011 as manuscript BJ20110260 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110260

Biochemical

  Journal Immediate Publication. Published on 08 Apr 2011 as manuscript BJ20110260 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110260

  Publication. Published on 08 Apr 2011 as manuscript BJ20110260 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110260 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited ϵ Table 2

  Publication. Published on 08 Apr 2011 as manuscript BJ20110260 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110260A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Publication. Published on 08 Apr 2011 as manuscript BJ20110260 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110260 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Nucleotide sequence and map of the LEE20-275 LEE1 regulatory region fragment A. The figure shows the base sequence of the LEE20-275 fragment, carrying the LEE1 P1 promoter, flanked by upstream EcoRI and downstream HindIII restriction sites. The sequence is numbered 1-120, starting with the first base of the cloned LEE1 regulatory region. The P1 promoter -35 and -10 hexamer elements are boxed and the transcript start site is indicated by a bent arrow. Mutations in the -35 and -10 promoter elements that cause reduction of P1 activity are indicated by vertical arrows. B. The figure shows the base sequence of the LEE20-275 fragment highlighting the sequence determinants of the P1A promoter. The P1A promoter -35 and -10 elements are boxed and the transcript start site is indicated by a bent arrow. The locations of the 88C, 89G, 90C and InsT (71-72) mutations that reduce P1A activity are indicated, together with the 64G and ǻ71T 'up' mutations.

Figure 2 . 2

 22 Figure 2. In vitro run-off transcription and potassium permanganate footprinting analysesA. The figure shows an autoradiogram of an analysis by gel electrophoresis of 32 P-labelled RNA transcripts made by RNA polymerase holoenzyme from DNA fragments carrying the starting LEE20-275 sequence (lanes 1-3), a derivative with the 98C mutation (lanes 4-6), or a derivative with the 64G and 98C mutations (lanes 7-9). The RNA polymerase concentration was: lanes 1, 4 and 7, no enzyme; lanes 2, 5 and 8, 200 nM; lanes 3, 6 and 9, 400 nM. The gel was calibrated with the pSR plasmid-encoded 108-base RNA-I transcript (lane 10) and Maxam-Gilbert sequence reactions (GA). The P1 and P1A transcripts are indicated by asterisks, and the proposed corresponding initiation sites are shown by bent arrows on the starting (WT) and mutant (64G 98C) sequences.B. The figure shows an autoradiogram that identifies the potassium permanganate sensitive sites in complexes of holo RNA polymerase with a DNA fragment carrying the starting LEE20-275 sequence (WT), a derivative with the 98C mutation, or a derivative with the 64G and 98C mutations. Lanes 1, 3 and 5 show the results after control incubations without RNA polymerase, while lanes 2, 4 and 6 show the analysis of samples with 50 nM RNA polymerase. The gel was calibrated using Maxam-Gilbert sequence reactions (GA) and relevant positions are indicated. Asterisks identify residues that display RNA polymerase-dependent reactivity to permanganate. Partial sequences of the WT and 64G 98C fragments are shown on the left and right sites respectively, with reactive sites marked by asterisks.

Table 3 . P1A promoter activity quantified by effects of the 88C substitution

 3 The table shows measured ȕ-galactosidase activities in cultures of E. coli strain M182 carrying pRW224 containing the LEE20-275 fragment and different mutations. Cultures were grown aerobically at 37°C in LB medium to an optical density of ~0.5 at 650 nm. Activities were measured in triplicate, giving a mean and standard deviation (SD). The central part of the table shows the fragment base sequence from position 62 to position 102, with the P1 promoter -10 and -35 hexamer elements grey shaded and P1A promoter -10 and -35 elements are underlined. Base substitutions in the different fragments are highlighted in boldface type whereas the ǻ71 deletion is shown by a dash. For each set of promoter fragments, the effect of the 88C mutation gives an estimate of P1A activity.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

This work was supported by a BBSRC project grant and by a Commonwealth PhD studentship to M.S.I. We are grateful to Kerry Hollands for help with the footprinting and transcription experiments.


Matthew Nicola J Rogers

James Simpson

Richard P Tuckett
email: r.p.tuckett@bham.ac.uk

Ken F Dunn

Colin J Latimer

Richard Zare

M J Simpson

Vacuum-UV negative photoion spectroscopy of CH4

Keywords: methane, ion-pair formation, vacuum-UV, absolute cross sections, quantum yields methane, ion-pair formation, vacuum-UV, absolute cross sections, quantum yields

Introduction

The interaction of electromagnetic radiation with the prototypical molecule, methane, is of fundamental interest because it is central to organic chemistry and abundant in our upper atmosphere. Much previous photochemical analysis has therefore been performed on the molecule. Being closed-shell and very stable, the lowest-lying excited electronic states of methane lie at high energies above the ground state, in the vacuum-UV region. As a consequence, much of the atmospheric photochemistry of methane is driven by intense solar atomic emission lines, such as Lyman-α radiation [START_REF] Wayne | Chemistry of Atmospheres[END_REF][START_REF] Cook | [END_REF].

Methane is a tetrahedral molecule with the electron configuration: (1a 1 ) 2 (2a 1 ) 2 (1t 2 ) 6 , and photoionisation has been studied by He I and He II Photoelectron spectroscopy [3][4][START_REF] Potts | Proc. R. Soc. Lond. A[END_REF][START_REF] Bieri | [END_REF][7][8][9]. The removal of the 1t 2 electron, at ca. 13 eV, is known to give rise to the triply-degenerate ground state of CH 4 + , and much detailed work has been carried out to study the nature of the Jahn-Teller distortion of the cation from tetrahedral symmetry [4,10,11]. The (2a 1 ) -1 band at ca. 22 eV gives rise to a long vibrational progression in the ν 1 (a-1 ) mode [8], and the (1a 1 ) -1 core excitation at ca. 290 eV has been studied with vibrational resolution, revealing a shorter ν 1 vibrational progression, reviewed in [12]. Recent experimental studies have been undertaken to investigate the non-Franck-Condon behaviour of this core photoexcitation [13,14]. Between the (2a 1 ) -1 and the (1a 1 ) -1 ionisation energies (IEs), weak satellite peaks have been observed in the photoelectron spectrum, which have been assigned as 'double-hole one-electron' states of CH 4 + [8]. The formation of doubly-excited states of methane by photoexcitation, which correspond to Rydberg states that converge on these satellite states of CH 4 + , have been investigated by dispersed fluorescence [15,16].

There have been many studies of the dissociation products following ionisation of CH 4, revealing the fragmentation dynamics of an energy-selected CH 4 + cation [7,9,[17][18][19], and detailed Lyman-α photofragmentation studies have also been undertaken [START_REF] Cook | [END_REF]20]. Total photoabsorption cross sections have been measured in the vacuum-UV range of 10-30 eV many times [21][22][23][24][25], with cross sections ranging from ca. (1 -5) x 10 -17 cm 2 .

In this paper we describe an experiment to detect anions following vacuum-UV excitation as a means to study the decay dynamics of electronically excited states of CH 4 due to ion-pair formation, generically described as AB → A -+ B + (+ neutrals). Absolute cross sections and quantum yields have been evaluated

for the anions observed. Photoion-pair formation has been detected for many diatomic and small polyatomic molecules [START_REF] Berkowitz | VUV and Soft X-Ray Photoionization[END_REF]. Dissociative ion-pair states can be accessed via direct ion-pair formation, i.e.

photoabsorption from the ground state directly to the ion-pair state, or via pre-dissociation following photoexcitation to an excited neutral state, often a Rydberg state. For the latter process to occur, the excited state must be formed at an energy greater than or equal to the asymptotic energy for ion-pair formation, and significant coupling between the two unperturbed wavefunctions is required [START_REF] Suits | [END_REF].

The formation of H -from CH 4 has previously been investigated in the energy range 12-27 eV by Mitsuke et al. [28,29]. In this paper, we confirm the H -efficiency curve features that they detected, and extend the range of detection to higher energy. However, there is a significant difference in our value for the absolute cross section for H -formation from that quoted by Mitsuke et al. We also report the first observation of the CH -and the CH 2 -anions from vacuum-UV photoexcitation of methane, and present absolute cross sections for formation of these anions.

Experimental

The ion pair apparatus has been described in detail previously [30]. Briefly, a direct jet of the gas under investigation is injected from a needle which orthogonally bisects the incident photon beam. The crossing point is positioned between two grids along the third Cartesian axis. A potential difference applied across these grids attracts negative ions towards a three-element electrostatic lens for focussing, and into a Hiden Analytical HAL IV triple quadrupole mass spectrometer (QMS) for mass selection. Anion detection is achieved by a channeltron electron multiplier. The apparatus and QMS were connected via a 1 mm diameter aperture, and were pumped by separate turbomolecular pumps, backed by a common rotary pump. Differential pumping enhances sensitivity by reducing the number of free electrons and secondary collisions in the QMS. All measurements were performed using vacuum-UV radiation from beamline 3.1 at the UK Daresbury Synchrotron Radiation Source (SRS), using a 1 m focal length Wadsworth monochromator to provide tunable radiation in the range 12-35 eV. These energies are available from the higher energy of two gratings mounted back-to-back in the monochromator [31]. The optimum resolution that can be obtained from this beamline is 0.05 nm, corresponding to ca. 0.01 eV at 15 eV. However, to enhance sensitivity, the spectra reported in this paper were recorded at a resolution of 0.25-0.60 nm. A 2 mm diameter, 300 mm long capillary light guide was used to connect the beamline to the experimental apparatus, providing the necessary differential pumping.

The base pressure of the apparatus was ca. 10 -7 mbar. The pressure was measured in the main chamber using an ionisation gauge, and the introduction of the sample gas to the system raised the pressure to ca. absolute cross sections of anion formation, was calibrated in a separate experiment relative to N 2 using a capacitance manometer [32]. Gas samples were obtained from Apollo Scientific with quoted purity of > 99.9 %, and were used without further purification.

Mass spectra were recorded to observe all anions produced from photoabsorption of the sample gas by exposure to white light, i.e. the grating is set to zero order to act as a mirror. The mass-to-charge ratio (m/z) of each peak in the mass spectrum was then defined, and the ion yield recorded as a function of photon energy. Once the peak positions were determined, the anion signal was recorded as a function of gas pressure over a typical range of ca. (0.5 -5.0) x 10 -5 mbar. Anions displaying a linear dependence with pressure can be attributed to ion-pair formation (i.e. AB → A -+ Β + (+ neutrals)), whereas those showing a non-linear pressure dependence cannot. The latter are likely to result from the two-step kinetic process of dissociative electron attachment (i.e. AB + hν → AB + + e -; AB + e -→ A -+ B), in which the rate of formation of A -is proportional to the square of the pressure of AB.

The yields of H -, CH -and CH 2 -all show a linear dependence with pressure. To determine their absolute cross sections, it is necessary to normalise the signals to the photon flux, the ring current, the gas pressure, the ionisation gauge sensitivity, and the relative mass sensitivity of the QMS to detection of the different anions. As in our previous studies on SF 5 CF 3 [33] and the CF 3 X series [34], we can write that:

( )         = frp SM k hν σ ( 1
)
where S is the detected signal normalised to unit time, f is the relative photon flux which effectively is a measure of the grating efficiency, r is the storage ring current, p is the sample gas pressure corrected for ionisation gauge sensitivity, and M is the relative mass sensitivity of the QMS. k is the normalisation constant. Normalisation to f, r and p is facile, but this is not the case for M. An extensive set of experiments was therefore performed to determine M as a function of (m/z), and is described in Section 2.1. The normalised signals can then be put on an absolute scale by calibration with the F -signals measured from SF 6 ((7 ±2) x 10 -21 cm 2 at 14.3 eV [35]) and from CF 4 ((1.25±0.25) x 10 -21 cm 2 at 13.9 eV [36]).

We note, however, that these cross section values from Mitsuke et al. are not strictly absolute, but are obtained indirectly from the signal of O -produced from O 2 at 17.3 eV, for which the absolute cross section is known [37]. Strictly, the values of the normalisation constants, k (F -/SF 6 ) and k (F -/CF 4 ), should be equivalent, but in fact they differ by a factor of 1.5. Given the number of corrections made to the anion signals in the two experiments, this discrepancy falls within a reasonable expected experimental uncertainty. The average value of k was then used in Equation (1) to determine the absolute cross sections, σ, with units of cm 2 , for production of H -, CH -and CH 2 -from CH 4 .

Mass Discrimination by the QMS

All quadrupole mass spectrometers exhibit an element of mass discrimination due to fringing fields, with a tendency to transmit heavier ions less efficiently [START_REF]Quadrupole Mass Spectrometry[END_REF]. To correct for this effect, the mass factor, M, has been determined by comparing the cation mass spectra of many polyatomic molecules in the QMS, following 70 eV electron impact ionisation, to actual mass spectra published in the electronic NIST database [START_REF]NIST Chemistry Webbook[END_REF]. It is assumed that any mass factors in the data presented in reference [START_REF]NIST Chemistry Webbook[END_REF] have been accommodated. M was calculated as a function of (m/z), see Figure 1, and was used to normalise the raw anions signals, as explained above. It can be seen that as (m/z) increases, the detection efficiency of the QMS decreases and a higher M value is required to correct this effect.

The zero-blast artefact [37], whereby all ions entering the quadrupole mass filter may be transmitted when the applied potentials are set to detect m/z 1, is not important in this CH 4 study because the H -signal is dominant. This effect, however, is important in the detection of the weak H -signal from CH 3 X (X = F, Cl, Br), where the spectra show significant contributions from the much stronger X -ion yields [START_REF] Rogers | [END_REF].

Thermochemistry

Our work determines appearance energies at 298 K (AE 298 ) for fragment anions from CH 4 , and these can be compared with calculated thermochemical values. Berkowitz has noted that for many polyatomic molecules, a calculated threshold energy is a lower limit to the experimental AE 298 value of an anion, when suitable assumptions are made about what the accompanying cation and neutral fragment(s) are [START_REF] Berkowitz | VUV and Soft X-Ray Photoionization[END_REF]. Furthermore, in comparing experimental AE values with calculated ∆ r H o 298 values of appropriate dissociation reactions, we are making two assumptions. Firstly, the dissociated fragments are not initially formed with thermal equilibrium, but rather are produced with conserved translational momentum relative to the centre of mass. Therefore a well-defined thermodynamic temperature cannot be allocated to the moieties and thermal corrections should be made [START_REF] Traeger | [END_REF]. Secondly, the effects of entropy have been disregarded, whereas for unimolecular dissociative reactions ∆ r G o 298 is always slightly more negative than ∆ r H o 298 because ∆n (the stoichiometric difference in the number of gas-phase molecules due to the reaction) and hence ∆ r S o 298 are both positive. Both of these effects are ignored in this study, which is deemed justifiable at the relatively modest resolution of the experiment.

Values for ∆ r H

Results and Discussion

Ion-Pair Spectra

The ion yields of H -, CH respectively. The resolution is 0.6 nm, corresponding to 0.07 eV at 12 eV and 0.28 eV at 24 eV. The H - signal is the most intense. Since all three anions show a linear dependence of signal with pressure, it is possible to determine the absolute cross sections for anion production. Using absolute, total vacuum-UV absorption cross section data from Au et al. [24], quantum yields for anion production can also be determined. Table 1 shows these data for the energies at which the H -, CH -and CH

CH 4 → CH -+ Η + + 2Η (8) CH 4 → CH 2 -+ Η 2 + ( 9
)
CH 4 → CH 2 -+ Η + + Η (10)
The thermodynamic thresholds can be used to infer the possible decay channels that give rise to each peak in the spectrum. The AE 298 of H -precedes the calculated ∆ r H o 298 value of reaction (2) by ca. 0.4 eV. This scientific impossibility could be accounted for by uncertainties in the thermochemistry, and/or by contributions from hotbands. However, it is clear that the first peak in the H -spectrum at ca. 15 eV can only arise from reaction [START_REF] Cook | [END_REF]. The double peak in the H -spectrum at ca. 21 and 22 eV probably corresponds to either or both decay channels shown in reactions (3) and ( 4). The third broad peak at ca. 28 eV has an approximate onset at ca. 23 eV, and may correspond to production of H -by reaction [START_REF] Potts | Proc. R. Soc. Lond. A[END_REF].

The CH -spectrum shows an onset at 22.5 ± 0.2 eV. A weak shoulder is observed up to ca. 27 eV, and a second, more prominent onset is apparent at this energy. The first onset could correspond to reaction [START_REF] Bieri | [END_REF] or (7), although we believe that the shoulder is probably an artefact of the spectrum, resulting from detection of the CH 6) and ( 7) from the onset of this peak would be reasonable, due to the formation of a new H 2 or H 2 + bond, respectively. An energy barrier resulting from forming these bonds is extremely likely, and thus their thermochemical thresholds would be expected to lie below the experimental onsets. The onset at 22.2 ± 0.2 eV in the CH 2 -yield agrees very closely with the enthalpy of reaction (10), but similarly the peak could correspond to reaction (9), with formation of

H 2 + .

The H - --

-yield from CH 4 We now consider the H -spectrum (Figure 2(a)) in more detail. The line positions and relative peak intensities agree well with those observed by Mitsuke et al. [28,29]. The first peak at ca. 15 eV, with onset at 13.3 ± 0.1 eV, has been assigned as a direct transition to an ion-pair state which dissociates to into H -( 1 S) + CH 3 + ( X ~ 1 A 1 ') [28]. We note that this onset lies well above the first adiabatic IE of methane, 12.61 eV [4,[START_REF] Potts | Proc. R. Soc. Lond. A[END_REF], and well below the second adiabatic IE, 22.39 eV [8]. The H -peak at 15 eV cannot, therefore, coincide with a Rydberg state of CH 4 , and its broad shape and slow onset indicate direct ion-pair formation [START_REF] Berkowitz | Photoabsorption, Photoionization and Photoelectron Spectroscopy[END_REF].

The region between 19.5-23.5 eV in Figure 2(a) contains fine structure that is shown in more detail in a higher-resolution spectrum (Figure 3). Peak positions are listed in Table 2. These features arise from an indirect process, in which an initially-excited Rydberg state predissociates into an ion-pair state. Thus the vacuum-UV radiation is probing the spectroscopic features of the Rydberg states. Vibrational structure is observed in three close-lying Rydberg states, and they have been assigned by Mitsuke et al. to the (2a 1 ) -1 (3p) 1 , (2a 1 ) -1 (4p) 1 and (2a 1 ) -1 (5p) 1 states [28,29]. These Rydberg states converge on the A ~ 2 A 1 state of CH 4 + , so the vibrational progressions observed should mimic closely any vibrational structure in the second photoelectron band of CH 4 . When an np Rydberg state is excited, the only allowed fundamental frequency in T d symmetry is the totally-symmetric ν 1 (a 1 ) C-H stretching mode. We observe a vibrational spacing in the (2a 1 ) -1 (4p) 1 Rydberg state, in which the vibrational structure is most clearly resolved, of 0.26 ± 0.02 eV or 2097 ± 160 cm -1 , to be compared with a value for neutral CH 4 of 2917 cm - 1 [42]. As expected, this matches the ν 1 vibrational spacing of CH 4 + A ~ 2 A 1 of 0.27 ± 0.01 eV, taken from the threshold photoelectron spectrum of this band (Figure 4, data listed in Table 3).

A Rydberg series of energy levels, E n , is described by the well-known formula: (11) where R H is the Rydberg constant, n is the principal quantum number of the orbital which runs to infinity upon convergence, and δ is its quantum defect. δ δ δ δ values for the 3p and 4p states are determined to be 0.62 ± 0.02 and 0.58 ± 0.02, to be compared with values of 0.67 and 0.60 from Mitsuke et al. [28].

F o r P e e r R e v i e w O n l y         - - = 2 ) ( δ n R IE E H n
There is some inconsistency in the literature whether to use the adiabatic or the vertical IE in such Rydberg calculations, and this choice can significantly affect the Rydberg assignments for large values of n, near the convergence limit. As the Rydberg formula determines the electronic series of states, E n (Equation ( 11)) should refer to the v"=0 → v'=0 transition from the ground state to the Rydberg state, and the adiabatic IE should be used in Equation (11). However, it is only possible to do this if vibrational structure is resolved in the spectrum and the transition to v'=0 is identifiable. In spectra that consist of many unresolved vibrational modes, it is more appropriate to use the vertical IE, because both the vertical Rydberg and vertical ionisation transitions will occur from v"=0 to the same value of v'. This makes the valid assumption that the geometry and vibrational spacing of the Rydberg and cation states are similar.

In determining the quantum defects of these Rydberg states of CH 4 , we have calculated all the v R → v + = v R transitions per Rydberg state, using the vibrationally-resolved term values in Tables 2 and3. 3 and4).

The values for the 3p and 4p

Between 23.5 and 33.0 eV, Figure 2(a) displays an extensive peak which has not previously been observed. This peak lies above the second adiabatic IE (22.39 eV [15]), but well below the next IE, ca.

290 eV , which corresponds to the (1a 1 ) -1 core ionisation. Thus, it features in an energy range absent of 'single-hole-one-electron' Rydberg states. In addition, the broad and structureless nature of the peak does provide some tangible evidence for a direct ion-pair process, and it could also result from a shape resonance. However, it seems more likely that this peak results from indirect ion-pair formation, -have all virtually disappeared at 32.1 eV. In addition, reactions ( 5), ( 8) and ( 10), which we believe to be the most likely routes for formation of these three anions at energies above 25 eV, all involve the production of neutral H atoms.

F

Absolute cross sections and quantum yields for anion formation

The absolute cross section for H -formation, 1.4 x 10 -22 cm 2 at the peak of the (2a 1 ) -1 (3p) 1 Rydberg state at 20.6 eV, is a factor of ca. 70 smaller than the value quoted by Mitsuke et al. at the slightly higher energy of 21.5 eV, the peak of the 4p Rydberg state [29]. Whilst the errors made in evaluating absolute cross sections are often underestimated in the literature, we do not believe that this major discrepancy can be explained by an accumulation of individual errors in the various correction factors described in Section 2. Overall, we believe that our cross sections are accurate to within a factor of 2-3. The corrections made by Mitsuke et al. to their signals to determine absolute σ values are not clear, and in particular it is not apparent whether they have applied any mass discrimination correction factor for detection of m/z 1 anions in their quadrupole mass spectrometer. We therefore measured the H -yield from C 2 H 6 and C 3 H 8 , obtained absolute cross sections in the manner described earlier, and compared our data with that of Mitsuke et al. for these larger saturated hydrocarbons [29]. Rather surprisingly, the results are in reasonable agreement [START_REF] Simpson | [END_REF]. For example, at the energy at which the cross section is a maximum, 18.9 eV, we determine σ (H -/C 2 H 6 ) to be 1.7 x 10 -21 cm 2 , to be compared with 2.2 ± 0.9 x 10 We note that in comparing cross sections for H -formation from CH 4 to F -formation from CF 4 , we might expect the H -cross sections to be smaller on electronegativity grounds because the C-H bonds are polarised C δ--H δ+ , whereas the C-F bonds are polarised C δ+ -F δ- [3]. Assuming that the cross section for F -/CF 4 at 13.9 eV, 1.25 x 10 -21 cm 2 [36], is correct, and indeed our data are calibrated to this value (Section 2), then it is surprising that the value of the cross section for H -/CH 4 from Mitsuke et al. is eight times greater than that for F -/CF 4 , whereas our value is nine times smaller. We have also observed this trend in cross sections (i.e. σ (F -) > σ (H -)) for larger hydrocarbons and their perfluorinated equivalents;

C 2 H 6 (C 2 F 6 ), C 3 H 8 (C 3 F 8 ) and C 2 H 4 (C 2 F 4 ). In each case, in the range 10-25 eV the maximum value of the F -cross section is a factor of 2-18 times greater than the maximum value for H -formation [START_REF] Simpson | [END_REF].

These arguments provide some evidence, and give confidence to our smaller value of σ (H -/CH 4 ) shown in Figure 2(a).

Using our cross section values and total absorption cross sections from (e,2e) spectroscopy [24], the absolute quantum yields for H -, CH -and CH 2 -formation can be calculated. They take values in the range 1-5 x 10 -6 (Table 1). These values are of the same order of magniture as those obtained in our earlier studies on CF 3 X (X = Cl,Br,I) [34] and SF 5 CF 3 [33].

Threshold Photoelectron Spectrum of the second band of CH 4

The TPES of the second band of CH 4 , ionisation to CH 4 + A ~ 2 A 1 (Figure 2(d), expanded in Figure 4), was recorded at the Swiss Light Source, Paul Scherrer Institute Villigen, Switzerland, using an iPEPICO spectrometer [44]. The photon resolution was 0.004 eV and the step size 0.002 eV. A single progression is observed in ν 1 , the totally symmetric C-H stretching mode of a 1 symmetry, which peaks at v=1. This observation is independent of whether peak intensities or areas are measured. This spectrum has been recorded before by several groups [START_REF] Bieri | [END_REF][7][8][9], but never with an experimental resolution as good as 4 meV. Our peak positions and separations (Table 3) are in excellent agreement with those determined by Carlsson Göthe et al. from He II photoelectron spectroscopy [8], and we can unambiguously confirm their tentative observation that the linewidth increases as the vibrational quantum number in the ν 1 mode increases. At a resolution of 4 meV, our signal-to-noise ratio is not good enough to determine peak positions or widths ). We therefore determine lifetimes of 21, 12, 8 and 6 fs, corresponding to dissociation rates of 4.8 x 10 13 , 8.3 x 10 13 , 1.2 x 10 14 and 1.7 x 10 14 s -1 , for v = 0, 1, 2 and 3, respectively.

for v ≥ 4,
We note that the lifetimes are lower limits and the dissociation rates are upper limits, because it is assumed that all the peak broadening is due to the lifetime effect. In particular, we have ignored any effects due to instrumental resolution and rotational fine structure. It seems very unlikely that the broadening is spectroscopic, and not dynamic, in nature, possibly due to unresolved vibrational polyads at higher energy caused by Coriolis and Fermi interactions, because these should be partially resolvable with a photon resolution of 0.004 eV.

Threshold photoelectron photoion coincidence measurements have shown that the

Conclusions

Absolute cross sections and quantum yields for production of H The assignment of this peak to v=0 is not definite, so the vibrational numbering of this progression is not certain. [START_REF]NIST Chemistry Webbook[END_REF]. At each (m/z) value, the % yield from NIST is divided by the % yield from our spectrum, and the data are normalised to unity at m/z 69 (i.e. CF 3 + ). The squares show the data points, the solid line shows the best fit to a third order polynomial. Progression (a) has been assigned as the (2a 1 ) -1 (3p) 1 Rydberg state, (b) as the (2a 1 ) -1 (4p) 1 Rydberg state, and (c) the (2a 1 ) -1 (5p) 1 Rydberg state.

  mbar. The sensitivity of the ionisation gauge to CH 4 , SF 6 and CF 4 , essential for determination of

  -and CH 2 -from CH 4 in the range 12-35 eV are shown in Figures 2(a) -2(c),

  transition is not categorically defined. We note that the same vibrational intensity distribution is not expected for the H - ---yield produced by indirect ion-pair formation through the np Rydberg series as a photoelectron spectrum of the CH 4 + A ~ 2 A 1 state; the former is an indirect twostep process, the latter is a direct one-step process. Indeed, a different intensity distribution is observed in the Rydberg 3p and 4p series (Figures

Figure 1 :

 1 Figure 1: Graph to determine the relative mass sensitivity of the Hiden Analytical HAL IV quadrupole mass spectrometer as a function of (m/z). Sample gases included CF 4 , SF 6 , CF 3 SF 5 , CH 3 F, CH 3 Cl, CH 3 Br, CH 2 Cl 2 , CF 2 Cl 2 , CFCl 3 , C 2 F 4 and c-C 5 F 8 . The mass spectrum of each sample was measured with 70 eV electron impact ionisation, and compared with the NIST spectrum[START_REF]NIST Chemistry Webbook[END_REF]. At each (m/z) value, the % yield from NIST is divided by the % yield from our spectrum, and the data are normalised to unity at m/z 69 (i.e. CF 3 + ). The squares show the data points, the solid line shows the best fit to a third order polynomial.

Figure 2 :

 2 Figure 2: (a)-(c) Cross sections for H -, CH -and CH 2 -production following vacuum-UV photoexcitation of CH 4 . Ion yields were measured between 12 and 35 eV with a step size of 0.1 eV and a wavelength resolution of 0.6 nm. Solid arrows show energies of thermochemical thresholds calculated for reactions (2)-(10), respectively. (d) Threshold photoelectron spectrum of CH 4 measured at a resolution of 0.004 eV using the imaging photoelectron photoion coincidence spectrometer at the Swiss Light Source[44]. The step size is 0.002 eV.

Figure 3 :

 3 Figure 3: High resolution H -scan between 19.5 and 23.5 eV. The step size is 0.02 eV and the resolution is 0.25 nm, ca. 0.09 eV. The three progressions (a)-(c), have been assigned by Mitsuke et al.[28,29] as vibrational progressions in ν 1 (a 1 ) within different Rydberg states converging on the à 2 A 1 state of CH 4 + .

Figure 4 : 23 Figure 4 Page 23 of 23 URL

 423423 Figure 4: Expansion of Figure 2(d). Threshold photoelectron spectrum of the second band of CH 4 , ionisation to CH 4 + A ~ 2 A 1 recorded with a step size of 0.002 eV and a resolution of 0.004 eV.

  o 298 of relevant ion-pair reactions were calculated using literature values for enthalpies of formation (∆ f H o 298 in kJ mol -1 ): CH 4 = -74.9, H = 218.0 (both Ref. 42); H -= 145, CH -= 477, CH 2

				-=
	327, H + = 1530, H 2	+ = 1488, CH + = 1619, CH 2	+ = 1386 and CH 3	+ = 1098 (all Ref. 43).

  As described earlier, we are not distinguishing a reaction enthalpy from a reaction energy). They take the following values;13.66, 18.91, 19.06, 23.58, 21.58, 23.40, 26.10, 19.59 and 22.28 eV.

	The arrows in Figures 2(a)-2(c) show the calculated ∆ r H o 298 values for ion-pair dissociation reactions (2)
	-(10). (CH 4 → H -+ CH 3 +	(2)
	CH 4 → H -+ CH 2	+ + H	(3)
	CH 4 → H -+ CH + + H 2 F CH 4 → H -+ CH + + 2H o r CH 4 → CH -+ H + + H 2	(4) (5) (6)
	CH 4 → CH -+ Η 2	P + + Η	(7)
			e
			e r
			R
			e
			v i e
			w
	to CH 4 O n l + A ~ 2 A 1 , recorded with the imaging photoelectron photoion coincidence (iPEPICO) spectrometer at the Swiss Light Source, Paul Scherrer Institute Villigen, Switzerland [44]. There is no similarity between this spectrum and any of the y
	three anion yields over this energy range. In addition to the pressure test, this is further evidence that the
	anions are not formed by dissociative electron attachment, but by ion-pair dissociation.
	4.1.1 Appearance energies and thermochemical thresholds
				7

2 -anions have maximum intensity; 20.6, 29.3 and 24.9 eV, respectively. A very weak signal was detected at m/z 15 (CH 3 -), but the signal-to-noise ratio was poor, and the features appeared to mimic those in the CH 2 spectrum. It is difficult to differentiate weak signals between anions in the QMS that are only one m/z unit apart, and this spectrum was therefore discarded. Figure 2(d) shows the vibrationally-resolved threshold photoelectron spectrum (TPES) of the second band of CH 4 , i.e. ionisation

  many close, unresolved 'doubly-excited Rydberg states' that converge to a doubly-excited IE of methane. Doubly-excited states of methane have been observed by dispersed fluorescence in the 25-35 eV energy range by Kato et al.[15]. The states converge to the 'double-hole one-electron' states of CH 4 + , observed as satellites in the photoelectron spectrum by Carlsson Göthe et al. [8]. Doubly-excited states were observed as a broad peak at ca. 29 eV in the study of Kato et al., which gave rise to fluorescence from atomic hydrogen. They were assigned as Rydberg states converging on the (1t 2 ) -2 (3a 1 ) 1 state of CH 4 + at 32.1 eV, that produce excited H atoms via predissociation [8]. Furthermore, it is possible that the broad peaks in the CH -and the CH 2 -spectra (Figures 2(b) and 2(c)) at ca. 29 and 25 eV, respectively, also arise from indirect ion-pair formation, resulting from different ionpair states crossing these same doubly-excited Rydberg states. We note that the signals of H -, CH -and CH 2

	following predissociation of
	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	10

  -21 cm 2 from Mitsuke et al. For H -/C 3 H 8 , at the peak energy of 18.7 eV, we determine a cross section of 3.4 x 10 -21 cm 2 , to be compared with the value from Mitsuke et al. of 1.6 ± 1.0 x 10 -21 cm 2 . It appears, therefore, that the

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

detection of m/z 1 anions is not the reason, per se, for the anomalously high value for σ (H -/CH 4 ) of

Mitsuke et al.

  although data up to v = 14 are quoted by Carlsson Göthe et al. Since our experimental resolution is much narrower than the peak widths, deconvolution of the experimental resolution is not necessary, and we directly determine full width at half maximum (FWHM) values of 31, 52, 82 and 99 meV for v = 0, 1, 2 and 3, respectively. These values are roughly a factor of two smaller than those

	modelled by Carlsson Göthe et al., and approximately correspond to the linewidths of the four vibrational
	levels of CH 4	+ A ~ 2 A 1 (ν 1
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y

Table 1 .

 1 The CH -and CH 2 -spectra are observed here for the first time. Whilst the relative yield of H -is very similar to that observed in an earlier study by Mitsuke et al.[28,29], our cross section values are a factor of ca. 70 smaller than those quoted earlier. This discrepancy remains unresolved. The H -peaks are attributed to both direct and indirect ion-pair formation, whereas the CH - and CH 2 -peaks probably arise from an indirect process, caused by ion-pair states crossing with doublyexcited Rydberg states. The threshold photoelectron spectrum of the second band of CH 4 , ionisation to CH 4 + A ~ 2 A 1 at 22.40 eV, has been recorded with a resolution of 0.004 eV. The widths of the peaks observed for v = 0-3 in the ν 1 vibrational ladder increase with v. They are the first direct measurement of a lower limit to the lifetime, and hence an upper limit to the unimolecular dissociation rate of these levels, AE 298 values, and absolute cross sections and quantum yields for production of anions from vacuum-UV photodissociation of CH 4 . Φ anion , calculated from total vacuum-UV absorption cross sections of CH 4 taken from Ref. 24.

	F r o dissociative electron attachment. into fragment ions.
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	13

-, CH -and CH 2 -from CH 4 over the energy range 12-35 eV have been determined. The signals of all three ions display a linear dependence with pressure, showing that they arise from an ion-pair mechanism and not from the multi-step process of b Cross section for ion-pair formation at the peak maximum. c Energy of peak maximum, at which σ anion (max) and Φ anion are taken. d Quantum yields for anion formation,

Table 2 .

 2 Vibrational progressions observed in the yield of H -from CH 4 (Figure3).

	ν 1 vibrational	Progression (a)	Progression (b)	Progression (c)
		state	E / eV	(∆E / eV)	E / eV	(∆E / eV)	E / eV	(∆E / eV)
	v =	0	20.00		21.24		22.06 a
				(0.28)		(0.28)		(0.26)
		1	20.28		21.52		22.32
				(0.24)		(0.24)		(0.28)
		2	20.52		21.76		22.60
				(0.24)		(0.24)
		3	20.76		22.00
						(0.24)
		4			22.24
	a

Table 3 .

 3 Full Width at Half Maximum (FWHM) values for vibrational peaks in the second photoelectron band of CH 4 , ionisation to CH 4 + A ~ 2 A 1 (Figure4).

			Molecular Physics	Page 18 of 23
		ν 1 vibrational state	E / eV	(∆E / eV)	FWHM / meV
	v =	0	22.40		31 ± 3
				(0.27)
		1	22.67		52 ± 5
				(0.26)
		2	22.93		82 ± 8
				(0.25)
		3	23.18		99 ± 10

URL: http://mc.manuscriptcentral.com/tandf/tmph

Acknowledgments

We thank Dr David Shaw (Manager of beamline 3.1) for help with recording the anion spectra at the Daresbury synchrotron source. We thank Drs Andras Bodi and Melanie Johnson (SLS) and Ms Jonelle Harvey (University of Birmingham) for help with recording the TPES of CH 4 on beamline X04DB of the Swiss Light Source. The collaboration between the groups in Birmingham and Belfast was partially funded by EPSRC Network Grant No. GR/N26234/01. The STFC and EU are thanked for the provision of beamtime at the Daresbury synchrotron and the Swiss Light Source, respectively.