Substitutions in Region 2.4 of σ70 Allow Recognition of the σS-Dependent aidB Promoter*

The strict dependence of transcription from the aidB promoter (PaidB) on the EσS form of RNA polymerase is because of the presence of a C nucleotide as the first residue of the –10 promoter sequence (–12C), which does not allow an open complex formation by Eσ70. In this report, σ70 mutants carrying either the Q437H or the T440I single amino acid substitutions, which allow –12C recognition by σ70, were tested for their ability to carry out transcription from PaidB. The Gln-437 and Thr-440 residues are located in region 2.4 of σ70 and correspond to Gln-152 and Glu-155 in σS. Interestingly, the Q437H mutant of σ70, but not T440I, was able to promote an open complex formation and to initiate transcription at PaidB. In contrast to T440I, a T440E mutant was proficient in carrying out transcription from PaidB. No σ70 mutant displayed significantly increased interaction with a PaidB mutant in which the –12C was substituted by a T (PaidB(C12T)), which is also efficiently recognized by wild type σ70. The effect of the T440E mutation suggests that the corresponding Glu-155 residue in σS might be involved in –12C recognition. However, substitution to alanine of the Glu-155 residue, as well as of Gln-152, in the σS protein did not significantly affect EσS interaction with PaidB. Our results reiterate the importance of the –12C residue for σS-specific promoter recognition and strongly suggest that interaction with the –10 sequence and open complex formation are carried out by different determinants in the two σ factors.

The sigma () subunits are responsible for the sequencespecific binding, correct promoter recognition, and transcription initiation by bacterial RNA polymerase. Seven different subunits have been identified in Escherichia coli; 70 is the main subunit during active growth and 70 -associated RNA polymerase (E 70 ) carries out transcription from the majority of E. coli promoters. Alternative subunits (e.g. H , N ) direct transcription toward specific sets of genes, often in response to cellular stresses, by promoting RNA polymerase binding to promoter sequences that strongly diverge from 70 consensus (1)(2)(3). However, the alternative S subunit, the product of the rpoS gene and mainly expressed in stationary phase of growth, can recognize similar promoter sequences and thus initiate transcription from several 70 -dependent promoters (4). Recognition of similar promoter sequences by 70 and S is reflected by their strong similarity in the DNA binding domains (5) and would be consistent with the need of the cell to continue expressing housekeeping genes in physiological conditions that simultaneously result in S accumulation and inhibition of 70 -dependent transcription, such as accumulation of intracellular guanosine tetraphosphate concentrations because of amino acid starvation (6 -8). In particular, the alignment of known E S -dependent promoters points to a high similarity of the Ϫ10 sequence with the canonical TATAAT sequence for 70 (9 -12), whereas no apparent conservation of the Ϫ35 sequence is evident. Likewise, the search for an optimal promoter for E S in vitro using the systematic evolution of ligands by exponential enrichment procedure has led to the proposal of CTATA(c/ a)T as the Ϫ10 consensus sequence for E S (13). This approach also resulted in the identification of TTGACA, i.e. the consensus Ϫ35 promoter element for E 70 , as the optimal Ϫ35 sequence for E S . These results suggest that promoter selectivity between 70 and S might be determined by different tolerance toward deviations from a common consensus sequence. In addition, factors independent of DNA sequence such as intracellular salt concentration, degree of DNA supercoiling, the guanosine tetraphosphate alarmone, and modulation by transacting regulatory proteins would help determine selective promoter recognition (14 -18). A sequence feature important for S -specific promoter recognition is a C nucleotide immediately upstream of the Ϫ10 promoter element (CTATA(c/a)T) and conventionally placed at the Ϫ13 position relative to the transcription start (Ϫ13C) (13, 19 -22). Site-directed mutagenesis of the S -dependent osmE and csiD promoters have confirmed the importance of the Ϫ13C element for efficient S -dependent transcription (11,23). Although the Ϫ13C nucleotide is an important determinant for E S -dependent transcription at the aidB promoter, (24) the major determinant for E S selectivity is the presence of a C nucleotide as first nucleotide of the Ϫ10 hexamer (Ϫ12C); indeed, its substitution to a T, canonical for the Ϫ10 element 70 -dependent promoters, results in loss of specific aidB promoter recognition by E S (24). The presence of a C as the first nucleotide of the Ϫ10 sequence is also responsible for specific recognition by S at the osmE (23) as well at the csgB and sprE2 promoters (33). Thus, although 70 displays a strict requirement for a thymidine as the first nucleotide of the Ϫ10 sequence, S appears to be able to recognize either pyrimidine.
The amino acids contacting the Ϫ10 promoter element are located in region 2.4 of 70 (25)(26)(27)(28)(29)(30), and the DNA-protein in-teraction appears to involve numerous residues between the 425 and 451 positions (31). Suppression genetics studies have identified two mutations, which allow recognition by 70 of a C nucleotide at position Ϫ12 at mutant lac and ant promoters (25,27). Both mutations are single amino acid substitutions at position 437 (a glutamine to a histidine, Q437H) and at position 440 (a threonine to an isoleucine, T440I). In region 2.4 of S , the amino acids at the corresponding positions are Gln-152 and Glu-155. Interestingly, both these residues have recently been implicated in S interaction with the Ϫ13C element at the osmE and fic promoters (32).
In this work we have investigated the ability of mutant 70 proteins at residues Gln-437 and Thr-440 to recognize a C as first nucleotide of the Ϫ10 element in the context of the strictly S -dependent aidB promoter. We show that both the Q437H and the T440E mutations allow efficient transcription initiation from the aidB promoter by E 70 through improved recognition of the C nucleotide at position Ϫ12 (first nucleotide in the Ϫ10 promoter element). However, substitutions of the corresponding Gln-152 and Glu-155 amino acids in S did not affect aidB promoter recognition, thus suggesting that interaction with the Ϫ10 promoter element might be carried out by different determinants in either S or 70 .
Protein Purification and Holoenzyme Reconstitution-Core enzyme and factors were purified as described previously (32,34,35); proteins appeared to be pure from contaminants as determined by denaturing protein gel electrophoresis (data not shown). Reconstitution of active holoenzymes for the different experiments was achieved by incubating the core enzyme and either S or 70 (either wild type or mutant) at a 1:4 ratio (to ensure saturation of the core enzyme) for 30 min at 37°C. For the competition experiment in a bandshift assay, the 70 : S :core ratio was reduced to 1, and reconstitution time was reduced to 10 min. The reconstituted holoenzymes were diluted at room temperature in K-glu200 buffer (40 mM HEPES, pH 8.0, 10 mM magnesium chloride, 200 mM potassium glutamate, 4 mM dithiothreitol, and 500 g/ml bovine serum albumin) prior to their use for in vitro experiments. Dithiothreitol was omitted from the buffer for permanganate (KMnO 4 ) reactivity experiments.
Binding and Transcription Assays-Single-round transcription assays were performed on derivatives of the supercoiled plasmid pJCDO1 (37) carrying either PaidB or its PaidB (C12T) mutant derivative, as described in Ref. 33. Plasmids (3 nM) and reconstituted RNA polymerase holoenzymes (50 nM) were incubated for 15 min at 37°C in K-glu200 buffer to allow the RNA polymerase-promoter complex formation. Elongation was started by the addition of a prewarmed mixture containing nucleotides and heparin (final concentrations were 500 M ATP, GTP, and CTP; 30 M UTP; 0.5 Ci of [␣-32 P]UTP; and 500 g/ml heparin) to the template-polymerase mix and allowed to proceed for 10 min at 37°C. Reactions were stopped by the addition of 10 l of loading buffer (formamide containing 20 mM EDTA, xylene cyanol, and bromphenol blue). After heating to 65°C, samples were loaded on 7% polyacrylamide sequencing gels. Reaction products from PaidB were quantified using a PhosphorImager (Molecular Dynamics) and normalized to the standard RNA-I product after background subtraction.
For gel mobility shift assays, the reconstituted holoenzyme (7.5-50 nM) and the 180-bp promoter DNA fragments (1 nM) were incubated for 15 min at 37°C in K-glu200 buffer in a final reaction volume of 10 l. The reaction mixture was then loaded onto a native 5% polyacrylamide gel after the addition of 2.5 l of loading buffer (50% sucrose, 0.025% xylene cyanol, 0.025% bromphenol blue, and 150 g/ml heparin).
For DNase I footprinting, reconstituted RNA polymerase (100 nM) was incubated with either PaidB or PaidB (C12T) (4 nM) for 30 min at 37°C in K-glu200 buffer. Protein-free DNA samples were treated with 1 g/ml DNase I for 20 s, whereas the incubation was prolonged to 30 s in the presence of RNA polymerase. After the addition of loading buffer containing 150 g/ml heparin, the samples were separated on a 5% native polyacrylamide gel, and the bands corresponding to the RNA polymerase-promoter complexes were eluted from the gel, precipitated, and resuspended in EDTA (20 mM)-formamide buffer before being loaded on a 7% polyacrylamide sequencing gel.
KMnO 4 reactivity experiments were performed as described (36). Briefly, 50 nM RNA polymerase and promoters were incubated in K-glu200 (without dithiothreitol) for 15 min at 37°C; KMnO 4 was added to a final concentration of 10 mM, and the reaction was stopped after 30 s by adding 2-mercaptoethanol to a final concentration of 330 mM. The KMnO 4 -reactive bands were expressed as a percentage of the total labeled DNA loaded onto the gel.

RESULTS
In Vitro Transcription at the PaidB with RNA Polymerase Assembled with Mutant 70 Proteins-In a previous work, we have shown that the presence of a cytosine (Ϫ12C) as the first nucleotide of the aidB Ϫ10 promoter element (CATACT) is the main determinant for specific PaidB promoter recognition by S -RNA polymerase (E S ). Substitutions of the Ϫ12C to a thymine nucleotide results in a near-consensus Ϫ10 sequence for 70 (TATACT) and allows efficient promoter recognition by E 70 (33). Mutations in 70 have been described that allow transcription initiation from mutant lac and ant promoters in which the T at position Ϫ12 had been substituted to a C (Plac (T12C), Pant (T12C) ); these mutant promoters are no longer recognized by wild type 70 -RNA polymerase (E 70 ) (25,27). We tested whether these mutations would also confer the ability to carry out transcription at the aidB promoter, i.e. in a S -dependent promoter context. In addition to the already characterized Q437H and T440I mutants, we constructed a T440E mutant 70 protein, in which the threonine at position 440 of 70 was substituted to a glutamate, i.e. to the corresponding amino acid residue in S (Glu-155). The different forms of RNA polymerase assembled with the three 70 mutants were tested in experiments of in vitro transcription on supercoiled templates and compared with wild type E 70 and E S (Fig. 1A). Both the Q437H and T440E mutants were more efficient (roughly 4-fold, Fig. 1B) than wild type 70 in carrying out transcription from wild type PaidB; in contrast, the T440I mutation did not improve aidB transcription significantly (1.2fold). Stimulation of transcription initiation by the Q437H and T440E mutations appears to specifically depend on increased recognition of the Ϫ12C. Indeed, transcription from the mutant aidB promoter in which the Ϫ12C has been substituted to a T (PaidB (C12T) ) was carried out at roughly the same extent by wild type and mutant E 70 s (Fig. 1). Although the C to T mutation at position Ϫ12 also stimulated E S -dependent transcription, the E S :E 70 ratio in transcription initiation was reduced from 9-fold at PaidB to 3-fold at PaidB (C12T) (Fig. 1B), consistent with previous observations (33).
Gel Retardation and DNase I Protection Experiments-Transcription initiation is a complex process that takes place in at least three distinct steps, binding of RNA polymerase to the promoter sequence, formation of the so-called "open complex," and promoter escape. At the aidB promoter, interaction with E 70 is limited at the open complex formation step (24,33). Thus, we investigated the ability of the Q437H, T440E, and T440I 70 mutants to form heparin-resistant complexes and to promote open complex formation at PaidB. Fig. 2  polymerase were able to promote formation of heparin-resistant complexes with PaidB, in contrast to E 70 (WT) and E 70 (T440I) , and consistent with the results of the in vitro transcription experiments (Fig. 1A). As expected, both wild type and mutant E 70 s were able to promote efficient heparin-resistant complex formation at the PaidB( C12T ) promoter (Fig. 1B).
The heparin-resistant complexes were probed with DNase I to gather more detailed information on the specific RNA polymerase-promoter interactions. Consistent with the results of the previous assays, both the E 70 (Q437H) and the E 70 (T440E) forms of RNA polymerase, but not E 70 (WT) and E 70 (T440I) , were able to protect the aidB promoter region from DNase I attack (Fig. 3A). The extent and the pattern of protection by E 70 (Q437H) and E 70 (T440E) were similar to E S ; however, subtle differences in the pattern of DNase I-hypersensitive bands by E S and E 70 could be detected in the region around and immediately upstream from the Ϫ35 element. Binding by both forms of RNA polymerase results in the appearance of DNase I-hypersensitive bands in both the Ϫ35 to Ϫ39 and in the Ϫ46 to Ϫ50 regions of PaidB. However, although binding by E S results in DNase I hypersensitivity mainly at Ϫ36 and Ϫ46 positions, binding by E 70 results in even stronger hypersensitive bands at Ϫ38 and Ϫ48 (Fig. 3A, shown by arrows). An identical DNase I-hypersensitivity pattern in the Ϫ35 region is induced by both wild type and mutant E 70 s at the PaidB( C12T ) promoter (Fig. 3B), suggesting that both the Q437H and the T440E mutations specifically result in increased interaction with the Ϫ10 promoter region, without affecting E 70 -promoter contacts in the Ϫ35 region.
KMnO 4 Reactivity Experiments-To directly assess the effect of the 70 mutations on the open complex formation step of transcription initiation, we performed KMnO 4 reactivity experiments on the wild type and mutant E 70 s. The results clearly showed that both the Q437H and T440E mutations, but not T440I, allow E 70 to carry out promoter opening at PaidB (Fig.  4). The extension of the KMnO 4 -reactive region, which corresponds to the single-stranded DNA region induced by open complex formation by RNA polymerase, as well as its intensity (Fig. 4B) is very similar for both the E 70 mutants and for E S . The KMnO 4 reactivity induced by E 70 mutants encompasses the ϩ2 position, which defines an open complex fully competent for transcription initiation at PaidB (24,33). As expected, both wild type and mutant E 70 s were fully capable of open complex formation at PaidB( C12T ), consistent with the results of the in vitro transcription assays (Fig. 1).
Thus, our results showed that mutations in the Gln-437 and Thr-440 amino acid residues of 70 increase promoter recognition and open complex formation at PaidB, consistent with increased promoter recognition of Plac (T12C) and Pant (T12C) (25,27). Although Gln-437 also corresponds to a glutamine residue in S (Gln-152), the position corresponding to Thr-440 in S is a glutamic acid (Glu-155). Thus, the observation that the T440E, but not the T440I, mutation allows PaidB recognition by E 70 would suggest that Glu-155 is directly involved in the interaction with the Ϫ12C residue by ⌭ S . To address this possibility, we performed KMnO 4 reactivity experiments with RNA polymerase assembled with a S mutant in which the glutamic acid at position 155 had been substituted to an alanine (E155A). In addition, a S Q152A mutant was also tested. As shown in Fig. 4, neither S mutant was affected in open complex formation at either PaidB or PaidB( C12T ). To test whether either the E155A or the Q152A mutations could affect steps in transcription initiation other than open complex formation, we performed in vitro transcription experiments on supercoiled templates. The results clearly showed that both mutant S proteins could carry out transcription from the aidB promoter when assembled into RNA polymerase at least as efficiently as wild type S (Fig. 5).
Competition Assays between E S and Mutant E 70 -In a previous report, we have shown that although the substitution of the Ϫ12C to a T nucleotide in PaidB restores an almost perfect Ϫ10 promoter element sequence for E 70 and leads to a dramatic increase of E 70 affinity for the aidB( C12T ) promoter, E 70 is outcompeted by E S in direct competition assays because of higher affinity by E S for S -specific determinants in the aidB promoter outside the Ϫ10 sequence (i.e. Ϫ13C, displaced TG motif, possibly sequences in the Ϫ35 region) (33). Thus, if the Q437H and T440E mutations specifically affect interaction with the Ϫ10 element of the aidB promoter, we would expect that RNA polymerase assembled with the mutant 70 s would still be disfavored in competition assays with E S for binding to PaidB. In contrast, if the mutations result in a more generalized increase of binding affinity to weak promoters, the mutant forms of E 70 might be able to outcompete E S . Thus, we determined relative affinity of the mutant E 70 (Q437H) and the E 70 (T440E) forms of RNA polymerase, as well as of E 70 (WT) , for wild type PaidB, measured as their ability to compete with E S for the promoter. Competition experiments were performed as gel retardation assays in the presence of heparin using equal concentrations of 70 , S , and core RNA polymerase mixed together prior to the addition of the promoter DNA. As shown in Fig. 6, the E S -and E 70 -PaidB complexes run with different electrophoretic mobility in a gel retardation assay. At the concentrations used in our assays, no E 70 -PaidB complex was detectable (Fig. 6, lanes 2-4); in contrast, both E 70 (Q437H) and E 70 (T440E) were able to form a complex with PaidB at low RNA polymerase concentrations (Fig. 6, lanes 6 and 10). Apparent higher affinity for the aidB promoter by the E 70 mutant forms at low concentrations of RNA polymerase is likely to depend on the higher affinity of 70 for the core RNA polymerase (39), which would result in more efficient assembly of E 70 than of E S . However, at S concentrations allowing efficient binding of core RNA polymerase, the E S form of RNA polymerase is clearly favored over both E 70 mutants (Fig. 6, lanes 8 and 12). DISCUSSION In this report, we have investigated the interaction between the S -dependent PaidB and three 70 mutants in region 2.4, i.e. the 70 protein domain involved in sequence-specific interaction with the Ϫ10 promoter element. Two of these mutants, namely 70 (Q437H) and 70 (T440I) , had already been described as able to restore transcription initiation from mutants of the lac and ant 70 -dependent promoters in which the first nucleotide of the Ϫ10 promoter element had been changed from a T (part of the TATAAT consensus sequence for 70 ) to a C (Plac (T12C) and Pant (T12C) ). The T to C substitution at Ϫ12 completely abolishes transcription initiation by wild type E 70 (25,27,29). At the aidB promoter, selective promoter recognition by S depends on the presence of a C at the Ϫ12 position, i.e. as first nucleotide of the Ϫ10 promoter element (CATACT), which does not allow open complex formation by E 70 ; substitution of the Ϫ12C to a T results in increased promoter recognition by 70 and loss of S -specificity (33). We show that 70 (Q437H) , but not 70 (T440I) , was able to efficiently initiate transcription at PaidB (Fig. 1). The Q437H substitution led to an almost 4-fold stimulation in transcription initiation in vitro, compared with a 1.2-fold stimulation by T440I (Fig. 1). The lack of increase in transcription initiation by the T440I mutant at the aidB promoter might depend upon its intrinsic lower affinity for promoters carrying at Ϫ12C in comparison to 70 (Q437H) ; indeed, although the experiments were performed in different genetic systems, the Q437H mutation appears to induce a more drastic stimulation of transcription from both Plac (T12C) and Pant (T12C) mutant promoters (25,27). However, substitution of threonine at position 440 to glutamic acid resulted in a clear increase in transcription from PaidB, similar to the effect of the Q437H mutation (Fig. 1). Thus, we could confirm the observations by Waldburger et al. (27) and Siegele et al. (25) that both Gln-437 and Thr-440 residues of 70 have an inhibitory effect on the recognition of a C residue as the first nucleotide of the Ϫ10 promoter element. The mutants do not appear to be affected in recognition of PaidB (C12T) , in which the C at position Ϫ12 has been substituted to a T, suggesting that the Gln-437 and Thr-440 residues do not play an essential role in the interaction with the Ϫ10 element, consistent with the results of extensive mutagenic analysis of 70 region 2.4 (31). Interestingly, a mutagenic analysis of regions 2.4 and 3.0 has shown that substitution of the glutamine at position 437 to an alanine (Q437A) improves the recognition of mutants in the synthetic extended Ϫ10 KAB-TG promoter in which either nucleotide of the TG motif had been changed to a C (37). Thus, it appears that a possible function of the Gln-437 residue could be to modulate 70 interaction with the Ϫ10 promoter region. At the aidB promoter, both the Q437H and the T440E mutations result in increased formation of a heparin-resistant RNA polymerase-promoter DNA complex, as determined by gel retardation assays (Fig. 2), and in increased open complex formation, as determined with KMnO 4 reactivity assays (Fig.  4). The mutations appear to specifically increase RNA polymerase interaction with the Ϫ10 region, without affecting binding to the Ϫ35 sequence and to the upstream promoter elements (Fig. 3). Despite their increased ability in transcription initiation and in open complex formation, both E 70 (Q437H) and E 70 (T440E) show lower relative binding affinity to PaidB than E S , as determined by direct competition experiments in gel retardation assays (Fig. 6), probably because of the presence of FIG. 6. A, competition experiment in gel mobility shift assay by E S (WT) and E 70 (either wild type or mutant derivatives) at P aidB . A typical bandshift assay in the presence of heparin (two repetitions) is shown. The core enzyme and both factors were reconstituted for 5 min at a ratio of 1:1:1 before RNA polymerase-promoter complex formation (12 min S -specific binding determinants (e.g. Ϫ13C, displaced TG motif (33)) that are not recognized by the E 70 mutants. Our results confirm the importance of a C nucleotide at the Ϫ12 position, i.e. as first nucleotide of the Ϫ10 promoter element, for the specific promoter recognition by S , at least at a subset of S -dependent promoters (roughly 20% of total (33)); both its substitution to a T (Figs. 1-4 (33)) and mutations in 70 leading to increased recognition of Ϫ12C result in a significant loss of specific promoter recognition by E S . Thus, S would appear to be "color blind", i.e. would not be able to distinguish between C and T at position Ϫ12, in contrast to 70 .
The observation that the T440E, but not the T440I, mutation leads to increased transcription initiation and open complex formation at PaidB would suggest that the glutamic acid residue could play a direct role in an interaction with the Ϫ12C nucleotide. Indeed, the residue corresponding to Thr-440 in S , which is able to recognize a C as first element of the Ϫ10 promoter, is a glutamic acid (Glu-155). In contrast, Gln-437 is also conserved in S (Gln-152), despite its apparent inhibitory effect on Ϫ12C recognition. However, substitution of the glutamic acid at position 155 of S to an alanine does not affect either transcription initiation or open complex formation by E S at PaidB (Figs. 4 and 5), strongly suggesting that the Glu-155 residue is not involved in interaction with Ϫ12C. Thus, it appears that interaction with the same promoter element might be carried out by different determinants in S and in 70 . Indeed, a recent report (32) proposes that at the osmE promoter both Gln-152 and Glu-155 residues are involved in the recognition of the Ϫ13C, an additional S -specific promoter feature present at roughly 70% S -dependent promoters (11,12,33), rather than the first nucleotide of the Ϫ10 promoter element.
At the aidB promoter, the Ϫ13C promoter element is necessary for optimal transcription in vivo and contributes to open complex formation by E S (24,33). However, neither the Q152A nor the E155A mutations affect transcription initiation at either PaidB or PaidB (C12T) (Figs. 4 and 5) thus suggesting that neither the Gln-152 nor Glu-155 residues play a major role in recognition of the Ϫ13C element at these promoters. This observation is in contrast with the role of the Gln-152 and Glu-155 residues in Ϫ13C recognition at the osmE (32). Thus, it is possible that different promoter elements and amino acid determinants in S might play different roles at distinct subsets of S -dependent promoters. For instance, the Ϫ35 promoter element appears to be dispensable at most S -dependent promoters, but it is essential at osmE (23,32). At a subset of S -dependent promoters responding to osmotic shock, the socalled GCGG motif in the Ϫ35 area acts as an additional promoter determinant for E S (38). Thus, it could be possible that the promoter context defines the strength of interactions with the different promoter elements (Ϫ10 sequence, Ϫ13C, TG motif, Ϫ35 sequence, GCGG motif, UP element, etc.) and, consequently, the importance of the distinct determinants in the S protein for promoter recognition and transcription initiation.