Promoter recognition and discrimination by EsigmaS RNA polymerase

Molecular Microbiology (2001) 42(4), 939–954

Promoter recognition and discrimination by EsS RNA polymerase Tamas Gaal,1 Wilma Ross,1 Shawn T. Estrem,1 Lam H. Nguyen,2 Richard R. Burgess2 and Richard L. Gourse1* 1 Department of Bacteriology, University of Wisconsin, Madison WI 53706, USA. 2 Department of Oncology, University of Wisconsin, McArdle Lab, Madison WI 53706, USA. Summary Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sS (EsS), and it is known that there is some overlap between the promoters recognized by EsS and by the major E. coli holoenzyme (Es70), the sequence elements responsible for promoter recognition by EsS are not well understood. To define the DNA sequences recognized best by EsS in vitro, we started with random DNA and enriched for EsS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EsS contained the known consensus elements (210 and 235 hexamers) for recognition by Es70. Using genetic and biochemical approaches, we show that EsS and Es70 do not achieve specificity through ‘best fit’ to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EsS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Es70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EsS versus Es70 thus presents an alternative solution to the problem of promoter selectivity.

Introduction Escherichia coli RNA polymerase (RNAP) consists of b, b’, s, v and two a subunits. The core enzyme (a2bb’v) is capable of transcription elongation, whereas s is required for specific promoter recognition and transcription initiation. Escherichia coli has seven different s subunits: Accepted 6 September, 2000. *For correspondence. E-mail rgourse@ bact.wisc.edu; Tel. (11) 608 262 9813; Fax (11) 608 262 9865.

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s70 (encoded by rpoD ), s54 (rpoN ), s32 (rpoH ), sS (rpoS ), sE (rpoE ), s28 (rpoF ) and sFecI ( fecI ), each responsible for directing RNAP to a different set of promoters, and, in so doing, changing the pattern of gene expression (Gross et al., 1998; Ishihama, 2000). Es70, the holoenzyme responsible for transcription of the majority of genes in E. coli, recognizes three DNA sequence elements (all sequences provided below are for the non-template strand, 50 to 30 ). The 210 element, consensus TATAAT, is centred approximately 10 bp preceding the transcription start site and is recognized by regions 2.3 and 2.4 of s70. The 235 element, consensus TTGACA, is 16–19 bp upstream from the 210 element and interacts with region 4.2 of s70 (Gross et al., 1998). The UP element, an AT-rich region upstream of the 235 hexamer, is recognized by the C-terminal domains of the a subunits (Ross et al., 1993; Blatter et al., 1994). In addition, some Es70 promoters contain an extension of the 210 hexamer, TGTGn, immediately upstream of the 210 motif, that is recognized by region 2.5 of s70 (Barne et al., 1997; Burr et al., 2000). Generally, the more of these elements present in a particular promoter and the more similar their sequences are to the consensus sequences, the better the binding by RNAP (Gross et al., 1998; Ross et al., 1998). The synthesis, accumulation, and activity of sS (also referred to as s38 or KatF) are controlled by a number of positive and negative regulatory systems (Hengge-Aronis, 1996a; Ishihama, 2000). As a result, sS accumulates in the cell at the beginning of stationary phase and directs RNAP to transcribe stationary phase-specific genes (Mulvey and Loewen, 1989; Nguyen et al., 1993). In addition, the presence of specific transcription factors, high intracellular osmolarity, and/or negative supercoiling can favour transcription by EsS at certain promoters (Hengge-Aronis et al., 1991; Kusano et al., 1996; Hengge-Aronis, 1996b; Ishihama, 2000). As sS is involved in responses to osmotic and oxidative stress, it is sometimes considered a master regulator of stress responses (Hengge-Aronis, 1996b). EsS is also required for virulence in a number of pathogenic bacteria (Beltrametti et al., 1999; Suh et al., 1999). Although promoters recognized by EsS have been compiled, they do not display a distinctive consensus sequence (Espinosa-Urgel et al., 1996; Ishihama, 2000). It has been suggested that EsS recognizes 210 elements similar, but not identical, to the consensus

940 T. Gaal et al. recognized by Es70 (Espinosa-Urgel et al., 1996; Lee and Gralla, 2001). It has also been suggested that a 235 consensus sequence for EsS, if it exists at all, is different from the consensus element utilized by Es70, for example, curved DNA with no specific 235 sequence (Espinosa-Urgel et al., 1996), CC instead of TT at the upstream end of the 235 element (Wise et al., 1996) or CTGCAA as a 235 element (Bohannon et al., 1991; Vicente et al., 1991; Ballesteros et al., 1998). Therefore, the consensus DNA sequence for recognition by EsS is still unclear. We chose an in vitro approach to determine the sequences recognized best by EsS, reasoning that understanding the biochemical behaviour of the enzyme in the absence of other factors, and then integrating these data with the extensive information about EsS obtained in vivo, might facilitate understanding of the behaviour of the enzyme in the complex environment of the cell. We generated a DNA library containing every possible sequence variant in the s recognition regions and used EsS to select those sequences that bound preferentially in vitro. The 210 and 235 hexamers obtained were identical to those recognized by Es70, raising an interesting problem about promoter selectivity of the two holoenzymes. Based on genetic and biochemical approaches, we show that the two forms of holoenzyme prefer the same consensus hexamers for transcription, and that specificity can be achieved by differential tolerance of the enzyme for deviations from the consensus. This model is consistent with the observation that many stationary phase-specific genes are transcribed in vitro by both Es70 and EsS (Tanaka et al., 1993; Tanaka et al., 1995), and that sS shows considerable sequence homology to s70 in the regions responsible for specific promoter recognition, 2.3, 2.4, 2.5 and 4.2 (Lonetto et al., 1992), but it differs conceptually from the standard model for promoter recognition in which each holoenzyme has its own discrete

Fig. 1. A. Steps in the in vitro selection process. See text for details. B. Electrophoretic mobility-shift illustrating that EsS-promoter complexes migrate to a different position than EsS-promoter complexes containing the same 32P-labelled DNA fragment.

DNA recognition elements responsible for transcribing discrete sets of promoters.

Results Rationale and experimental design Footprinting experiments with an EsS-specific promoter, bolA1, indicated previously that EsS protects about 70 bp of promoter sequence (from about 257 to 117), similar in length and position to that protected by Es70 (Nguyen and Burgess, 1997). Based on this, and on the similarity in the amino acid sequences of the two s factors in the regions responsible for DNA binding (regions 2.3 –2.5, and 4.2), we decided to sample a population of DNA sequences containing all possible sequences from 238 to 12. The number of potential variants for 40 bp of DNA is about 440 (¼ 1024) different DNA sequences, far too many to test in vivo. Therefore, to test all possible recognition sequences for optimal EsS binding, we used an in vitro selection procedure with DNA fragments containing randomized promoter sequences. Similar approaches have been utilized previously to identify optimal binding sequences for numerous nucleic acid binding proteins (Blackwell and Weintraub, 1990; Pollock and Treisman, 1990; Tuerk and Gold, 1990; Wright et al., 1991; Estrem et al., 1998; 1999). One copy of each possible 40 bp sequence variant would generate about 1 kg of DNA, far exceeding what can be manipulated experimentally even in vitro. Therefore, we performed the in vitro selection in two steps, randomizing a 20 bp segment to generate libraries containing at least one copy of 420 (,1012) different sequences in each step. In the first step, a selection was performed with promoter fragments randomized from 218 to 12. This was followed by a second selection with promoter fragments randomized from 238 to 219 in the context of the selected 218 to 12 sequence. Highly purified EsS, containing no detectable s70 or other s factors (Nguyen et al., 1993), was added to the library of DNA fragments under limiting conditions so that only 0.5 –5% of the DNA fragments formed complexes with RNAP. After separation of the complexes from free DNA on a non-denaturing polyacrylamide gel, DNA from the bound fraction was eluted, PCR-amplified and the procedure was repeated for multiple cycles to enrich for the best binding sequences (Fig. 1A). As EsS-DNA complexes migrated to a location different from Es70-DNA complexes (Fig. 1B), contamination with DNA fragments bound by undetectable levels of Es70 (if present) would be minimized at each cycle in the enrichment. We note that our binding assay selects for sequences resulting in the maximal rate of open complex formation, but these sequences need not be optimal for Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Promoter recognition and discrimination by Ess RNAP each step leading to individual intermediates in the transcription initiation pathway.

Selection of the 210 promoter region by EsS To identify EsS promoter determinants in the region containing the 210 element, EsS was incubated with a promoter fragment library containing randomized sequences from 218 to 12 and bolA1 promoter sequences from 254 to 219 and from 13 to 116 (210 rnd; Fig. 2A). After 17 cycles of selection by EsS, 16 DNA fragments were cloned and sequenced (see Experimental procedures ). A close match to the 11 bp sequence TGTGCTATA(C/A)T was present on each of 12 promoter fragments (Fig. 2B and C; the other four fragments contained large deletions and probably were selected for artifactual reasons described in Experimental procedures ). The 11-mer sequence (e.g. in 210 sel 1) showed limited similarity to the bolA1 promoter but contained the Es70-like 210 hexamers, TATAAT or TATACT (Fig. 2). The 11-mer also contained the consensus ‘extended 2100 motif TGTG, that is found

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upstream of the 210 hexamer in a subset of E. coli promoters (Burr et al., 2000), and a C between the extended 210 motif and the 210 hexamer. The other 9 bp of the randomized region differed among the selected promoter fragments, indicating the promoters were of independent origin. The position of the selected 11-mer was relatively constant within the randomized region (from 217 to 27 in eight cases, from 216 to 26 in three cases, and from 218 to 28 in one case). These data suggest that sequences outside the randomized region contribute to positioning EsS on the DNA and, therefore, have an important role in recognition by EsS.

Selection of the 235 promoter region by EsS To identify promoter determinants for EsS binding upstream of 218, a second selection was performed with a fragment library (235 rnd; Fig. 3A) containing randomized basepairs from 238 to 219. Flanking sequences in the starting population derived from the 210 sel #1 promoter (218 to 116; Fig. 2) and from the

Fig. 2. 210 region selection. A. Non-template strand DNA sequences of the bolA1 promoter, the starting DNA population (210 rnd ) and 210 sel #1 [the first sequence in (B)]. The randomized region in 210 rnd (218 to 1 2) is indicated by ‘NNN…’. The Es70 consensus elements (Gross et al., 1998; Burr et al., 2000) are indicated. B. Sequences from the randomized region of 12 DNA fragments cloned after 17 cycles of selection with EsS in vitro. Sequences were aligned by identities in the 210 region. C. Frequency of bases at each position in the 12 cloned promoters from (B). Numbers in bold indicate positions at which the same base was found in at least 10 out of the 12 promoters or at which either of two bases were found in all 12 promoters.

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Fig. 3. 235 region selection. A. Non-template strand DNA sequences of the starting DNA population (235 rnd ) and 235 sel #1 [the first sequence in (B)]. The starting population (235 rnd ) contained the ‘SUB’ upstream sequence (254 to 219; Rao et al., 1994) and the 210 region from 210 sel #1. Randomized region (238 to 219) is indicated by ‘NNN…’. The Es70 consensus elements (Gross et al., 1998; Burr et al., 2000) are indicated. B. Sequences from the randomized region of 19 DNA fragments after 17 cycles of selection with EsS in vitro. C. Frequency of bases at each position in the 19 cloned promoters from (B). Numbers in bold indicate positions at which the same base was found in at least 16 out of the 19 fragments.

‘SUB’ promoter (254 to 239; Rao et al., 1994). The 254 to 239 SUB sequence does not contain RNAP recognition motifs (Rao et al., 1994) and was introduced to eliminate interference from 235-like sequences upstream of position 238 in bolA1 (Nguyen et al., 1993). Twenty-two promoter fragments were cloned and sequenced after 17 cycles of selection. Nineteen promoter fragments of the correct length contained the sequence CTTGACA from positions 236 to 230, exactly 17 bp upstream from the 210 hexamer (Fig. 3B and C); the other three fragments contained large insertions and probably were selected for artifactual reasons, as described in Experimental procedures ). In addition, there was a strong preference for A residues at positions 229 and 228 and a lesser preference for T at 227. Positions 238, 237 and 226 to 219 were different from clone to clone, confirming that the selected promoters were of independent origin. The selected 235 promoter region showed no similarity to

the same region of the bolA1 promoter, but it contained the consensus 235 hexamer for recognition by Es70. Characterization of the selected promoters in vitro To analyse the effects of the selected sequences on RNAP binding and transcription, promoters were constructed containing the selected 238 to 219 and 218 to 12 regions (either individually or combined together). To facilitate direct comparison with the bolA1 promoter, each construct contained sequences from bolA1 at all positions outside the randomized regions (Fig. 4A). For simplicity, these constructs are referred to as the ‘in vitro selected’ promoters, including 210 con (containing the selected 218 to 1 2 region from 210 sel #1; Fig. 2), 235 con (containing the selected 238 to 219 region from 235 sel #1; Fig. 3) and full con (containing both selected regions). To verify that the selection resulted in DNA sequences Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Promoter recognition and discrimination by Ess RNAP

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Fig. 4. In vitro transcription. A. DNA sequences (254 to 116) of the promoters analysed. Sequences outside of the in vitro selected regions are identical in all four promoters and derive from bolA1. The fragments were inserted into a plasmid that contains a transcription terminator ,170 bp downstream of the transcription start site (11). B. Transcription from the four promoters with EsS. The transcripts from the four promoters are indicated as ‘test’. The RNA I transcript from the vector is also indicated. Reactions contained Transcription buffer (see Experimental procedures ), 100 mM KCl and approximately 1 nM RNAP. C. Transcription from the same templates as in (B) but with Es70 instead of EsS. EsS and Es70 were assembled by addition of fivefold excess of s to the same amount of core enzyme (see Experimental procedures ). The faint transcript noticeable above the test transcripts in the Es70 reactions originates from a weak promoter whose putative 235 hexamer derives from bolA1 sequences about 10– 13 bp upstream of the 235 hexamer responsible for the major transcript (Nguyen et al., 1993). D. Transcription of the selected promoters at different KCl concentrations in vitro under otherwise identical reaction conditions as in (B) and (C). The y-axis is the ratio of specific product (from either the 210 con, 235 con or full con promoter) resulting from transcription by EsS vs. Es70. E. Ratio of transcription by EsS to Es70 at different K-glutamate concentrations.

that bound EsS better than bolA1, we compared the affinities of the DNA fragments for EsS under conditions identical to those used for the selection (see Experimental procedures ). The three selected promoters bound EsS substantially better than bolA1 (data not shown), confirming that the selection worked as expected. The relative order of binding was full con . 235 con . 210 con .. bolA1. To assess whether the increase in EsS binding by the selected promoters relative to bolA1 resulted in increased transcription, the activities of the three selected promoters were compared with the activity of the bolA1 promoter in vitro. When transcribed by EsS, all three selected promoters were much more active than bolA1, and the 235 con promoter was the strongest, in buffers containing 100–800 mM K-glutamate or 10–400 mM KCl (Fig. 4B and data not shown). The difference in activity between bolA1 and the selected Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

promoters was less dramatic at high RNAP concentrations (data not shown). As the selected promoters also contained the known consensus sequences for Es70 recognition, we tested their transcription by Es70 in vitro under the same conditions used for EsS. The selected promoters were transcribed very efficiently by Es70, whereas transcription from the bolA1 promoter was hardly detectable (Fig. 4C). The selected promoters were transcribed similarly by both holoenzymes in buffers containing 10–400 mM KCl (Fig. 4D and data not shown) or 100– 800 mM K-glutamate (Fig. 4E and data not shown). In no case were the promoters transcribed more than about twofold better by EsS holoenzyme than by Es70. Therefore, in contrast to the situation for some osmotically regulated promoters that are transcribed only by EsS at high concentrations of K-glutamate (Ding et al., 1995; Kusano and Ishihama,

944 T. Gaal et al. 1997), the selected promoters did not exhibit a large preference for EsS at high osmolarity. Because EsS and Es70 were prepared by adding saturating amounts of s factor to aliquots of the same preparation of core enzyme, it is valid to compare the relative activities of EsS and Es70 on each of the promoters (see Experimental procedures ). For each selected promoter, the level of transcription by EsS and Es70 was very similar, although the three selected promoters differed in activity from each other. In contrast, the wild-type bolA1 promoter was transcribed substantially better by EsS than by Es70, whereas the plasmid-derived RNA I promoter was transcribed much better by Es70 than by EsS. We conclude that the in vitro selected promoters are transcribed much better than the bolA1 promoter in vitro, but they do not exhibit specificity for EsS over Es70.

Fig. 5. Transcription in vivo. A. The four promoters analysed in vitro (Fig. 4A) were fused to lacZ in single copy on the chromosome, and b-galactosidase activities were measured in exponential and stationary phases as described in Experimental procedures. The bolA1 promoter was measured in System I (see Experimental procedures ) to allow direct comparison with the other System I promoter – lacZ fusions, but bolA1 was also measured in System II (Inset, see Experimental procedures ) for more precise determination of the exponential/stationary phase ratio shown in (B). (C) and (D) were the same as (A) and (B) except the promoter– lacZ fusions were in an isogenic strain lacking rpoS.

Transcription from the full consensus promoter ( full con ) was always weaker than from the promoter containing only the consensus 235 region, 235 con (Fig. 4B and C). The full con promoter’s relative inactivity results from a defect in promoter escape (T. Gaal, R.L. Gourse and N. Shimamoto, unpublished). Characterization of the in vitro selected promoters in vivo We measured the activities of the bolA1, 210 con, 235 con and full con promoters as promoter –lacZ fusions to determine whether the relative activities observed in vitro were also observed in vivo, and also to determine whether the promoters displayed the increase in promoter activity in stationary phase characteristic of EsS-dependent promoters (Fig. 5). The relative activities of the promoters were consistent with the activities observed in vitro: the 235 con promoter was three to fivefold stronger than the 210 con or full con promoters, and all three were much stronger than the bolA1 promoter, both in exponential and in stationary phase (Fig. 5A and C). The bolA1 promoter fragment was also introduced into a lacZ fusion system that produces higher b-galactosidase activity and lower background (‘System II’; Simons et al., 1987; Rao et al., 1994), allowing a more accurate estimate of transcription from weak promoters (see Fig. 5A and C inset). bolA1 promoter activity increased about 18-fold in stationary phase relative to exponential phase (Fig. 5B). In contrast, there was less than a twofold increase in bolA1 promoter activity in stationary phase in a strain lacking rpoS (Fig. 5D), confirming previous reports that the bolA1 promoter is EsS-dependent (e.g. Lange and Hengge-Aronis, 1991). Although the 210 con, 235 con and full con promoters were selected for binding to EsS, they were transcribed efficiently even when EsS was not present (Fig. 5C), and their activities increased only slightly in stationary phase (Fig. 5D). As the 210 con, 235 con and full con promoters are transcribed very efficiently by both EsS and Es70 in vitro, and as the promoters contain the consensus hexamers recognized by Es70, we conclude that these promoters are transcribed by Es70 in exponential phase and are probably transcribed by either EsS or Es70 (or both) in stationary phase. [s70 is more abundant than sS, even in stationary phase (Jishage et al., 1996)]. The residual rpoS-independent small increase observed for all four promoters in stationary phase could derive from decreased competition for limiting Es70 when rRNA transcription drops in slow or non-growing cells (see Barker et al., 2001). Interactions of EsS and Es70 RNAPs with specific promoter positions The results from the in vitro selections described above Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Promoter recognition and discrimination by Ess RNAP suggested that both EsS and Es70 prefer the same 210, 235 and extended 210 consensus sequences. However, the identification of sequences in in vitro selection experiments does not imply that each selected position has the same relative importance to RNAP binding, as even small increments to binding will result in selection, nor do these experiments address whether EsS and Es70 interact with a particular promoter in the same manner. To address whether the two enzymes recognize the same promoter sequence differently, we used interference footprinting, a technique in which DNA fragments with modifications at different positions in the population can all be assayed for protein binding at the same time. Specifically, if a modification at a certain position interferes with RNAP binding, the promoter fragment containing that modification will be underrepresented in the bound population. Interference footprinting thus allows estimation of the relative contribution of individual positions in a promoter to RNAP binding (although of course the effects

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of every functional group or even every basepair are not tested by any one interference probe). We tested the effects of two different modifications that alter the major groove surfaces of thymine or adenine in DNA on binding by EsS and Es70. Uracil is identical to thymine except it lacks the C5 methyl group in the major groove (Devchand et al., 1993), whereas 7-deaza-7-nitroadenine (A*) introduces a bulky nitro group into the major groove (Min et al., 1996). dUTP, or dA*, was incorporated into promoter fragments at low frequency by polymerase chain reaction (PCR) (Ross et al., 2001). The effects of these substitutions on Es70 binding to the rrnB P1 promoter have been characterized previously (Ross et al., 2001). Preliminary experiments suggested that single base modifications in the full con promoter did not interfere enough with binding by either holoenzyme to alter the fraction bound in gel-shift assays (data not shown). Therefore, the 235 con and rrnB P1 promoters were used instead; these promoters bound both holoenzymes, but

Fig. 6. EsS and Es70 bind differently to the same promoter sequence. A. Interference footprints of the 235 con promoter with uracil substituted for thymine on average once per DNA fragment. The promoter fragment was labelled at the 30 -end of the nontemplate strand. Signals result from strand cleavage only at the sites of uracil substitution. Scans are shown above the gel images (grey, no RNAP; blue, EsS-bound complex; red, Es70-bound complex). The 210 and 235 hexamers are in upper case letters, and the thymine residues discussed in the text are indicated below the gel images. B. Interference footprints of the rrnB P1 promoter with A* substituted for adenine on average once per DNA fragment. The promoter fragment was labelled at the 30 -end of the nontemplate strand. Signals result from strand cleavage only at the sites of A* substitution. Colour coding used in the scans is the same as in (A). The adenine residues in the 210 hexamer discussed in the text are indicated below the gel images. For simplicity, the residues in the 210 hexamer are numbered as in the 235 con promoter shown in (A). Because the distance between the transcription start site and the 210 hexamer in rrnB P1 is 2 bp longer than in 235 con, ‘211’ and ‘28’ are actually at 213 and 210 in rrnB P1.

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946 T. Gaal et al. with lower affinity than the full con promoter, so that uracil or A* substitutions resulted in changes in the fraction of the DNA population bound by RNAP. Uracil incorporation at three non-template strand positions in the 235 con promoter (one in the 210 region at 212, and two in the 235 region at 234 and 235) reduced binding by both EsS and Es70 (Fig. 6A). Uracil at 212 virtually eliminated binding by both enzymes. In contrast, uracil at either 234 or 235 greatly reduced Es70 binding, but only moderately reduced EsS binding. Similar differential effects of uracil substitution at 234 and 235 on Es70 versus EsS binding were also observed on the rrnB P1 promoter (data not shown). These results suggested that even though both holoenzymes prefer the same promoter sequence, they interact with that promoter differently. The effects of A* substitutions for adenine in the nontemplate strand on Es70 and EsS binding to the rrnB P1 promoter are illustrated in Fig. 6B; qualitatively similar results were obtained for the 210 con and 235 con promoters (data not shown). Modification of either of two positions in the 210 hexamer reduced RNAP binding, whereas no effects were observed in the 235 region. Binding of the two holoenzymes was affected differentially

Fig. 7. Substitution of CC for TT at the first two positions in the 235 element differentially affects transcription by EsS versus Es70 both in vivo and in vitro. A. The activity of the CC235 con promoter was measured in exponential (light bars) and stationary phase (dark bars) as described in Fig. 5. Promoter activities (in Miller units) were measured from promoter– lacZ fusions (System II, see Experimental procedures ) in strains containing or lacking rpoS. The CC235 con promoter is like 235 con except that it has a CC substitution for TT at the first two positions in the 235 hexamer. A promoter fragment with endpoints of 240 to 12 was used to construct both the promoter– lacZ fusion and the plasmid template for in vitro transcription. b-Galactosidase activities from the strains used in Fig. 7 (RLG5852, RLG5857) should not be compared with those reported in Fig. 5, because the promoters assayed in Fig. 5 were in a different fusion system (see Experimental procedures ) and had different endpoints (254 to 116). B. Supercoiled plasmid pRLG5881containing the CC235 con promoter (240 to 12) was transcribed with either EsS or Es70 (approx. 1 nM) in vitro as described in the legend to Fig. 4.

at one of the 210 region positions, the highly conserved A211 in the 210 hexamer. At this position, A* decreased EsS binding by about 60% but decreased Es70 binding by only about 30%. As with the uracil interference experiments, the A* footprints indicate that EsS and Es70 bind differently to the same promoter sequence. The less than total inhibition observed from introduction of A* for the highly conserved A211 could be attributable to the fact that the N1 position on the base is most crucial for A211 function (Matlock and Heyduk, 2000), not the 6 and 7 positions on the base altered in A*. A* substitution at A29 had a smaller effect than at A211, and A* substitution at A28 had a larger effect than at A211, but in both cases the effects were similar for the two holoenzymes. The strong inhibition of RNAP binding by A* at 28 could be attributable to steric clash by introduction of the bulky nitro group, as typically mutations at 28 have less severe effects on RNAP interactions than at 211. Basis for promoter selectivity by EsS versus Es70 The similarity in the intrinsic promoter recognition properties of EsS and Es70 raises the issue of how some promoters could be almost entirely dependent on EsS. That is, what accounts for some promoters being transcribed only by EsS when both EsS and Es70 are present in stationary phase, and for these same promoters not being transcribed by Es70 in exponential phase? In this section, we propose a model to address these questions and then we provide experimental support for this proposal. Our data suggest that the preferred hexamer sequences might be identical for EsS and Es70. However, naturally occurring promoter sequences rarely match the consensus perfectly for a particular RNAP. Differences from the Es70 consensus sequence, in conjunction with the effects of specific activators and repressors, are responsible for differences in levels of transcription initiation from individual Es70 promoters. The interference footprints shown above indicate that certain modifications of the 210 and 235 hexamers affect binding by EsS and Es70 differently. Therefore, to explain differences in transcription by EsS versus Es70, we suggest that non-preferred basepairs in a promoter sequence might have different effects on recognition by the two enzymes. To test this general concept, we chose two positions in the 235 element for detailed examination. The choice of these positions was dictated by the results of our interference footprinting studies (Fig. 6A) indicating that the loss of the methyl groups at positions 234 and 235 affect discrimination by Es70 versus EsS and by previous genetic evidence suggesting that C substitutions at 234 and 235 result in specificity for EsS (Wise et al., 1996; Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Promoter recognition and discrimination by Ess RNAP Bordes et al., 2000). To confirm that the identities of 234 and 235 contribute to discrimination by Es70 versus EsS, we constructed a CC-35 con promoter (i.e. a 235 con promoter containing T-34C and T-35C substitutions) fused to lacZ. The promoter DNA fragment used in this fusion extended only to 240 to eliminate any contribution to activity in vivo from a weak Es70-dependent promoter upstream of the 235 hexamer that derives from bolA1 sequences (see Fig. 4C).

Fig. 8. Multiple substitutions at the first two positions in the 235 element differentially affect transcription by EsS or Es70 in vitro. Supercoiled plasmids containing the 235 con promoter (TT at positions 235 and 234) or the indicated variants at these positions were transcribed with either EsS or Es70 (approx. 4 nM ) in vitro as described in the legend for Fig. 4 and Experimental procedures. Promoter activity is plotted relative to that of the 235 con promoter for each enzyme. The 235 con promoter is transcribed similarly by EsS and Es70 under these buffer conditions (see Fig. 4). Transcription was measured at least three times from each promoter and is expressed relative to transcription of 235 con promoter. Differences in the relative levels of transcription were less than 15% between experiments. A. Transcription with EsS in vitro. B. Transcription with Es70 in vitro. C. Ratio of transcription with EsS/Es70 in vitro.

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CC235 con was considerably weaker than 235 con and displayed the characteristics of an EsS-specific promoter in vivo (Fig. 7A). Transcription from CC235 con greatly increased in stationary relative to exponential phase, and this increase was almost completely dependent on the presence of rpoS. (The promoter endpoints and the lacZ reporter system used in Fig. 7 differ from those used in Fig. 5. Thus, the absolute b-galactosidase activities in the two figures should not be compared directly (see also Experimental procedures ). We also tested transcription from CC235 con with EsS and Es70 in vitro (Fig. 7B). Consistent with the results obtained in vivo, CC235 con was preferentially utilized by EsS in vitro. EsS tolerated the CC substitution much better than did Es70 under every condition tested. To test whether EsS can tolerate only the CC substitution at 234 and 235 or whether other substitutions at these positions are tolerated as well in 235 con, we generated all possible single and some two basepair substitutions for the TT at 234 and 235, and we compared transcription from each mutant by EsS in vitro to that from 235 con (Fig. 8A). None of the mutant promoters was more active than 235 con, confirming that TT at these two positions is optimal for recognition by EsS. None of the substitutions decreased transcription by EsS more than about twofold. CC235 con was transcribed by EsS worse than most of these promoters, indicating that CCGACA (Wise et al., 1996) is not the consensus 235 hexamer for recognition by EsS. As the promoter with the single G substitution at 235 was transcribed as well as 235 con in vitro, but promoters with this substitution were not obtained in the in vitro selection, it is possible that the in vitro transcription assay is not sufficiently sensitive to detect the slight preference for the consensus T at this position, or there is an effect of promoter context in this case. In contrast to their small effects on transcription by EsS, the TG, AA, CC, or GG substitutions decreased transcription by Es70 as much as 10-fold, resulting in a six to 10-fold preference for EsS over Es70 (Fig. 8B and C). These results are consistent with the model that discrimination between EsS and Es70 is accomplished by differential tolerance of the two enzymes for specific deviations from the consensus sequence.

Discussion Es70 and EsS recognize the same consensus hexamer sequences We have studied the intrinsic promoter recognition properties of EsS using an in vitro selection to identify the DNA sequence determinants for EsS binding. In contrast to expectations based on previous proposals (see

948 T. Gaal et al. Introduction ), the binding sites selected by EsS contained the consensus hexamer sequences for binding Es70, and they initiated transcription at high levels by both holoenzymes. Many EsS-dependent promoters rely predominantly on recognition of the 210 region (e.g. Tanaka et al., 1995; Espinosa-Urgel et al., 1996; Colland et al., 1999; Lee and Gralla, 2001). However, the results of the in vitro selection and the extraordinary strength of the 235 con promoter, relative to the bolA1 promoter, when transcribed by EsS in vitro, suggest that the 235 element can also play a prominent role in recognition by EsS in some promoter contexts. In fact, some EsS-dependent promoters (e.g. osmE; Bordes et al., 2000) have almost consensus 235 regions and appear to be strongly dependent on 235 hexamer interactions. Differential tolerance of RNAP holoenzymes for deviations from the same consensus hexamers As both Es70 and EsS prefer the same consensus hexamer sequences, there must be a mechanism for preventing transcription from stationary phase-specific promoters by Es70 in exponential phase. (Preventing transcription from these promoters by EsS in exponential phase is accomplished by limiting expression of sS.) We suggest that holoenzyme specificity might rely, at least in part (see below), on tolerance of the two enzymes for different deviations from the same consensus sequences. This hypothesis is supported by interference footprinting studies showing that EsS and Es70 recognize the same promoter sequence differently (Fig. 6) and by the effects of certain promoter mutations constructed as a test of this concept (Figs. 7 and 8). We found that, in the context of the 235 con promoter, substitutions of TT to TG, CC, AA, or GG at the first two positions of the 235 hexamer resulted in transcription in vitro that was strongly dependent on EsS, in contrast to 235 con. These results are consistent with genetic studies on the proU promoter, in which TT to CC substitutions at the same positions strongly reduced transcription and made it dependent on rpoS in vivo, and on the osmY promoter, in which CC to TT substitutions decreased discrimination between EsS and Es70 in vivo (Wise et al., 1996). We did not evaluate the relative preferences of EsS and Es70 for each possible basepair at every position in the consensus hexamers. However, several results strongly suggest that, in addition to positions in the 235 hexamer, there are positions in the 210 hexamer at which interactions with the two RNAPs are differentially affected by the same base modification or base substitution. These include the results of our interference footprints (Fig. 6), the fact that the promoter differing from bolA1 only in the 210 region (210 con ) is recognized by EsS but has lost its

EsS specificity (Fig. 4), and recent studies showing there is differential binding of EsS and Es70 to fork-junction templates with substitutions in the 210 region (Lee and Gralla, 2001). At position 28, A and C were about equally represented in the sequences selected by EsS, whereas A is considered the consensus for Es70-dependent promoters. Although there are some differences between s70 and sS in the region predicted to interact with position 28 (region 2.3; Lonetto et al., 1992; Malhotra et al., 1996; Fenton et al., 2000), we suggest that the identity of 28 does not play a major role in discrimination between the two holoenzymes. We constructed promoters differing only by A or C at this position and found they were transcribed similarly by the two enzymes in vitro (data not shown), and previous reports suggest that both Es70 and EsS tolerate A or C at 28 (Oliphant and Struhl, 1988; Kolb et al., 1995; Tanaka et al., 1995; Espinosa-Urgel et al., 1996). Recognition of either A or C at 28 by both enzymes could potentially be attributable to an interaction with the same functional group on both bases, for example the amino group on position 6 of A, or 4 of C. Other potential contributions to differential promoter recognition by EsS and Es70 Recent studies have proposed that a C at position 213 (i.e. the position just upstream from the 210 hexamer) is preferred by EsS, and G213 is preferred by Es70 in certain contexts, for example in the csiD and osmY promoters (Becker and Hengge-Aronis, 2001) and in fork-junction templates (Lee and Gralla, 2001). The results of our in vitro selection strongly support the preference of EsS for C at 213. However, C213 is clearly not sufficient to discriminate against recognition by Es70 in the context of the in vitro selected promoters described here (Figs. 4 and 5). In addition, the rrn P1 promoters that are transcribed much more efficiently by Es70 than by EsS (data not shown) contain a C at this position. Residues in addition to the consensus hexamers and C-13 were selected by EsS in our in vitro binding experiments, including a cytosine at the upstream flank of the 235 hexamer (236), two adenines at the positions just downstream of the 235 hexamer (229 and 228) and the sequence TGTG at positions 214 to 217 (the extended 210 region); 236, 229, and 228 are not usually considered as being determinants of Es70 binding and, therefore, it is formally possible that they might contribute to specific EsS recognition. However, we consider it improbable that these basepairs, by themselves, are responsible for discrimination between EsS and Es70, as (i) C is also preferred by Es70 at 236 in the lac and rrnB P1 promoters (Reznikoff, 1976; Josaitis et al., 1990), and (ii) A to G substitutions for the A residues at Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Promoter recognition and discrimination by Ess RNAP 229 and 228, at least in the context of the 235 con promoter, did not affect activity substantially in vitro with either holoenzyme (data not shown). [We note, however, that an A-tract downstream of the 235 hexamer was identified as a requirement for efficient rRNA promoter activity by Chlamydia trachomatis RNAP (Tan et al., 1998)]. The interaction between the extended 210 motif (TGTG) and region 2.5 of s has been well documented as an important determinant of both EsS- and Es70-dependent transcription (Voskuil et al., 1995; Barne et al., 1997; Becker et al., 1999; Colland et al., 1999; Burr et al., 2000). A spacer length of 17 bp between the 210 and 235 hexamers was strongly favoured for recognition by EsS in our in vitro selection. We found that Es70 tolerated a 16 bp spacer in the context of the rrnB P1 promoter much better than EsS, but a 16 bp spacer reduced the activity of the full con promoter approximately the same for both holoenzymes (data not shown). Therefore, spacer-length could contribute to EsS versus Es70 discrimination at some promoters, but our preliminary studies indicate that the effect of spacer length on the two holoenzymes is contextdependent and complex. Finally, our hypothesis does not exclude a role for additional DNA-binding proteins in promoter discrimination by EsS and Es70. That is, expression of some promoters (although not those investigated here) is influenced by differential effects of transcription factors or nucleoidassociated proteins on the two holoenzymes (Arnqvist et al., 1994; Bouvier et al., 1998; Colland et al., 2000).

s determinants for differential recognition of the same consensus hexamers The similarity between the EsS and Es70 consensus sequences is consistent with the similarity of the motifs in the two s factors responsible for promoter recognition. Sixteen out of 28 amino acids in region 4.2 (residues 572 – 599 of s70) and 25 out of 40 amino acids in regions 2.3 and 2.4 (residues 417 –456 of s70) are identical in the two s factors (Lonetto et al., 1992), including several residues implicated in direct interactions with specific basepairs in the 210 and 235 hexamers (e.g. Q437 in region 2.4, and R584 and R588 in region 4.2; s70 numbering, Gross et al., 1998). Superimposed on the overall conservation between the two s factors in the regions responsible for DNA binding are discrete regions of divergence that could contribute to promoter discrimination. Two clusters of four amino acids in region 4.2 (residues 578 –581 and 591– 594 of s70) are virtually identical among s70 members from different species and among different sS members from different species, but these sequences differ between s70 and sS (Lonetto et al., 1992) and could potentially play a role in discrimination between deviations from the consensus Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

949

235 hexamer. K173 of sS has been implicated in recognition of C213 (Becker and Hengge-Aronis, 2001). The corresponding residue in s70 is different (E458), consistent with their different DNA recognition preferences. As interactions with the 210 element are complex, involving binding to both single-stranded and doublestranded DNA in multiple steps during the process of transcription initiation, it is more difficult with present information to predict specific amino acid residues in s70 and sS that might play a role in differential 210 region recognition. Promoters recognized by both EsS and Es70 The observed overlap in the sequences recognized by EsS and Es70 potentially allows some promoters to be transcribed by both holoenzymes under some conditions, and yet to be transcribed by only one or the other holoenzyme under other conditions. Even though a promoter might be transcribed by both enzymes under some conditions, changes in the environment that alter template geometry (e.g. superhelicity) or solute concentrations (e.g. anions or cations) might be sufficient to alter the ratio of transcription by the two holoenzymes (Ishihama, 2000). It has been reported that the two holoenzymes possess different tolerances for high concentrations of K-glutamate or trehalose, and that this favours specific promoter recognition by EsS (Ding et al., 1995; Kusano and Ishihama, 1997; Nguyen and Burgess, 1997). We compared the activities of EsS and Es70 on the bolA1, 210 con, 235 con, and full con promoters in buffers containing 100 –800 mM K-glutamate or 10–400 mM KCl (data not shown). As predicted from previous studies (Leirmo et al., 1987), the anion glutamate was less disruptive to RNAP –promoter interactions than chloride, so transcription by both holoenzymes was generally higher at the same cation concentration in glutamate buffers. Tolerance for high salt concentrations may contribute to the ability of certain promoters to be transcribed only by EsS (Ding et al., 1995; Kusano and Ishihama, 1997). However, the in vitro selected promoters described here are transcribed by both holoenzymes at high osmolarity; high salt does not result in holoenzyme specificity. The degree of negative supercoiling of the bacterial chromosome, on average, is somewhat lower in stationary phase than in exponential growth, and it has been suggested that reduced supercoiling may enhance transcription by EsS rather than by Es70 (Kusano et al., 1996). Our promoter selections were performed on DNA fragments (linking number ¼ 0), but the promoters were also transcribed efficiently by both holoenzymes on supercoiled plasmid templates (linking number ¼