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Nucleic Acids Research Pages 3584-3590  

The -16 region of Bacillus subtilis and other gram-positive bacterial promoters
Introduction
Materials And Methods
   Plasmids, enzymes and reagents
   Bacillus subtilis RNA polymerase purification
   In vitro transcription
   Quantitation by primer extension
   Compilation of promoter consensus sequences
Results
   Effect of promoter mutations on transcription by B.subtilis RNAP
   Effect of promoter mutations on transcription by E.coli RNAP
   Role of the -16 region in `strong' promoters
   Transcription from two promoters
   amyP2 transcription
   [sigma]A stimulation of transcription
   Analysis of B.subtilis promoter sequences
   Gram-positive bacterial promoter sequences
Discussion
Acknowledgements
References

The -16 region of Bacillus subtilis and other gram-positive bacterial promoters

The -16 region of Bacillus subtilis and other gram-positive bacterial promoters

Martin I. Voskuil, Glenn H. Chambliss*

Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706, USA

Received February 23, 1998; Revised and Accepted June 5, 1998

ABSTRACT

The Bacillus subtilis [alpha]-amylase promoter amyP contains an essential TGTG motif (-16 region) upstream of the -10 region. Mutations of this region significantly reduced in vitro promoter strength. A -15 G->C transversion eliminated transcription from amyP by both B.subtilis and Escherichia coli RNA polymerase (RNAP). A second [alpha]-amylase promoter (amyP2) also required the -16 region for function. To determine conserved sequences in promoters containing -16 region elements, sequences of 64 B.subtilis promoters with the second TG motif of the -16 region were aligned and analyzed. Unlike the E.coli class of `extended -10 promoters', with a similar TG motif but lacking a -35 region, the -16 region promoters contain highly conserved -35 regions. They also contain conserved An and Tn tracts upstream of the -35 region. In addition, we analyzed all available gram-positive bacterial promoter compilations to determine the generality of the -16 region. From this analysis, the -16 region TRTG motif (R = purine) appears to be a basic element found in a large portion of gram-positive bacterial promoters and is, in the case of at least the [alpha]-amylase promoters, necessary for transcription by the major form of B.subtilis and E.coli RNAP.

INTRODUCTION

[alpha]-Amylase is an extracelluar starch-degrading enzyme produced by the gram-positive bacterium Bacillus subtilis (1). The major form of B.subtilis RNAP, E[sigma]A, is thought to recognize amyP (2) even though there is only a 3/6 match to the consensus E[sigma]A promoters at both the -35 and -10 regions (Fig. 1). The E[sigma]A consensus promoter with the sequences TTGACA and TATAAT at the -35 and -10 regions respectively is identical to the consensus sequence recognized by the major form of Escherichia coli RNAP, E[sigma]70. The six bases of amyP that match the consensus promoter are the bases most often conserved in both B.subtilis and E.coli promoters (3,4). The spacer sequence of amyP is 17 bases, the average distance between the -35 and -10 region of B.subtilis and E.coli promoters.

Bacillus subtilis and E.coli promoters transcribed by either E[sigma]A or E[sigma]70 have several similarities: the conserved sequences in the -35 and -10 regions, the distance between the regions and the position of the transcription start site. Even so, it has been noted that many functional E.coli promoters, such as the lacUV5 promoter, are not transcribed by B.subtilis RNAP, while B.subtilis promoters normally function well in E.coli (5-7). Bacillus subtilis promoters contain several moderately conserved sequences that may be the key to whether or not the promoter is utilized in B.subtilis. These include A- and T-rich regions upstream of the -35 region and A residues just downstream of the -10 region (3). In addition to these sequences, a region ending 1 base upstream from the -10 region is conserved. The sequence 5[prime]-RTRTG-3[prime] was first found to be conserved in nine B.subtilis promoters and was termed the -16 region (8). Recently, a more comprehensive analysis of 142 promoters, all with experimentally determined transcription start sites, has confirmed that the -16 region (TRTG) is conserved (3). The TG dinucleotide motif, positioned 1 base upstream of the -10 region, was found in 45% of the B.subtilis promoters. The T was found in 52% and the G in 58% of promoters. The T and the R residues were also found to be correlated with the presence of the TG dinucleotide (3).

Although the -16 region is moderately conserved in B.subtilis, there is less evidence for conservation of these sequences in E.coli promoters (4). The TG motif, 1 base upstream of the -10 region, has been shown to play a vital role in several E.coli promoters, called `extended -10 promoters', including a derivative of the [lambda] Pre promoter (9), the galP1 promoter (10) and the cysG promoter (11). The `extended -10 promoters' lack an identifiable -35 region but are transcribed by E[sigma]70. These promoters appear to bypass the need for a -35 region with the TG motif (9,11,12). Point mutations in the TG motif of the [lambda] Pre, galP1 and cysG promoters reduced or eliminated promoter function (9-11). The TG motif introduced into the galPcon6 promoter (galPconTG) reduced the temperature requirement for open complex formation by 20°C compared with galPcon6 (13), indicating that the TG motif may be important in isomerization from a closed to an open complex in transcription initiation. The TG motif described in E.coli promoters appears to be analogous to part of the -16 region TRTG motif described in B.subtilis.


Figure 1. Sequences of the [alpha]-amylase wild-type and mutant promoter regions. The -35, -16 and -10 regions of amyP are underlined. The -35, -16 and -10 regions of amyP2 are overlined. The -16 regions of both promoters are also shown in bold. The start site of amyP and the two start sites of amyP2 transcription are indicated by * and ** respectively.

We have shown that mutations in the amyP -16 region, with the sequence TGTG, all had detrimental effects on the production of [alpha]-amylase. The G->T transversion at position -15 essentially eliminated [alpha]-amylase production in both B.subtilis and E.coli (2). In this report, we further define the role of the -16 region in amyP by examining the effects of mutations in the -16 region and in the -35 and -10 regions on the in vitro utilization of amyP. To confirm that the -16 region is an E[sigma]A promoter element, in vitro transcription reactions were performed with purified E[sigma]A supplemented with additional [sigma]A. In addition, B.subtilis promoter sequences containing the -16 region TG motif were analyzed to determine additional conserved regions of -16 region promoters. Promoters from several gram-positive bacteria were also analyzed.

MATERIALS AND METHODS

Plasmids, enzymes and reagents

Plasmid DNA was isolated from E.coli using the Magic Minipreps DNA Purification system (Promega Biotech) with the following change. Plasmid DNA purification resin, prepared as described (14), was substituted for Promega Magic Miniprep Resin. Plasmid DNA concentration and purity were determined by measuring absorbance at 260 and 280 nm. The percent DNA was calculated as described (15) using the equation %N = (11.16R - 6.32)/(2.16 - R), where R = A260/A280. DNA concentrations were adjusted accordingly.

Escherichia coli RNAP and all chemicals were purchased from Sigma Chemical Co. unless otherwise indicated. AMV reverse transcriptase was purchased from Promega Biotech.

Bacillus subtilis RNA polymerase purification

Bacillus subtilis E[sigma]A was purified as described (16) with the following modifications. NaCl was substituted for KCl in the TGED buffers (50 mM Tris-HCl, pH 8.0, 5-20% glycerol, 0.1 mM EDTA and 0.1 mM dithiothreitol). Cell pellets from the 1E51 protease-deficient strain of B.subtilis (Bacillus Genetic Stock Center) were washed twice with buffer 1 (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM EDTA, 10% glycerol and 1 M KCl) and once with TGED-20% glycerol. The cells were lysed by passing through a French press twice. Proteases were further removed from the crude extract by use of a hemoglobin slurry (17). The extract was passed through a heparin-agarose column followed by a DNA-cellulose column as prepared by Alberts and Herrick (18). Final purification was accomplished with a Sephacryl S-300 column (19).

Bacillus subtilis [sigma]A was overproduced in E.coli strain BL21/DE3 containing pCD2 (20). The [sigma]A was purified from inclusion bodies by renaturation with 6 M guanidine-HCl, batch elution from DEAE resin and final purification by HPLC quaternary amine ion exchange chromatography (Protien-Pak Q 8HR, 10 × 100 mm; Waters) (21).

In vitro transcription

In vitro transcription was performed on supercoiled DNA of the plasmids listed in Figure 1. The RNA products were quantitated by primer extension analysis. Reaction mixtures consisted of 75 ng (0.04 pmol) plasmid DNA, 1.8 pmol B.subtilis RNAP or 4.7 pmol E.coli RNAP (Sigma Chemical Co.) and binding buffer (40 mM Tris-HCl, pH 7.9, 125 mM KCl, 10 mM MgCl2, 20 mM DTT and 5% glycerol) in a final volume of 12 µl and were incubated on ice for 5 min. Transcription was initiated by addition of a 48 µl binding buffer solution containing 400 µM nucleotides and transcription was allowed to proceed for 10 min at 37°C. tRNA (10 µl, 0.4 mg/ml) was added and the reactions were stopped and extracted with 70 µl phenol, pH 8.0. Sodium acetate and ethanol were added to precipitate RNA from the 45 µl aqueous phase from each extraction, followed by a 70% ethanol wash. The RNA produced from in vitro transcription reactions was quantitated by primer extension analysis. This allowed the use of non-linear plasmid DNA and the intact [alpha]-amylase gene in in vitro transcription reactions. The supercoiled plasmid DNA system more closely resembles the in vivo conditions compared with a system using linearized DNA to generate run-off transcripts. This system also allowed the analysis of several mutants without the excess manipulation and mutagenesis that would have been necessary to insert restriction sites or terminators into each mutant.

Quantitation by primer extension

The precipitated RNA from in vitro transcription reactions was suspended with 3 µl buffer (50 mM Tris-HCl, pH 8.3, 60 mM NaCl and 10 mM dithiothreitol) and 2 µl RBS-3 primer (0.8 pmol 105 c.p.m. labeled and 5 pmol unlabeled). The 5[prime]-labeling of the RBS-3 primer was carried out as described (2). The primer and RNA were allowed to anneal for 15 min at 45°C, after which 7.75 µl 50 mM Tris-HCl, pH 8.3, 60 mM NaCl, 6 mM MgCl2, 375 µM dNTPs, 4 U AMV reverse transcriptase (Promega Biotech) and 1.2 µg actinomycin D were added. The extension reactions were incubated for 30 min at 45°C and stopped with 8 µl 98% formamide, 10 mM EDTA, pH 8.0, 0.025% xylene cyanol FF and bromophenol blue. Extension products were quantitated by PhosphorImager analysis performed on a Molecular Dynamics PhosphorImager. To establish that neither RNAP in the in vitro transcription reactions nor primer in the primer extension reactions was limiting, pAMY10 DNA from 25-125 ng (0.01-0.07 pmol) was assayed. The level of transcription corresponded directly to the amount of DNA, demonstrating a limiting amount of DNA and an excess of other components.

Compilation of promoter consensus sequences

A published table (3) of B.subtilis sequences was obtained from Dr John D.Helmann's World Wide Web page (URL http://www.bio.cornell.edu/microbio/helmann/helmann.html ). The 142 promoters with experimentally confirmed transcription start sites were divided into the 64 promoters containing the TG motif 1 base upstream of the -10 region and 78 promoters that lack the TG motif. Tables of the two promoter groups were aligned as published (3). Sequences from -83 to +7 for each group were analyzed. The frequency of occurrence of each of the four bases at each position was calculated and the percentage of the most frequent base was graphed. Bacillus subtilis promoter regions have an overall base composition of 35% A, 15% C, 18% G and 32% T (3). These frequencies were used to calculate the statistically significant conserved bases of the two promoter groups.

Figure 2. In vitro transcription of mutant [alpha]-amylase promoters with B.subtilis (a) and E.coli (b) RNAP. In vitro transcription was performed on supercoiled plasmid DNA (Table 1) and quantitated by PhosphorImager analysis of primer extension products. Lane pAMY10, wild-type amyP; pAK15, -15 G->C mutation; pAK75, -16 T->C mutation; pAK85, -17 G->T mutation; pAK86, -18 T->A mutation; pAK19, -19 T->A mutation; pCON, amyP with consensus -35 and -10 regions; pCON15, amyP with consensus -35 and -10 regions and the -15 G->C mutation.

RESULTS

Effect of promoter mutations on transcription by B.subtilis RNAP

All mutations in the -16 region reduced in vitro transcription from amyP. A G->C transversion at position -15 virtually eliminated promoter function, reducing in vitro transcription by 95%. Mutations at positions -18, -17 and -16 reduced amyP utilization to ~25% of the wild-type level. At position -19, 1 base upstream of the -16 region (pAK19), a T->A transversion had essentially no effect on amyP utilization (Fig. 2a and Table 1).

Table 1. Summary of in vitro transcription
Promoter/
Plasmid		 B.subtilis RNAP in vitro
RNA (relative amount)*		 E. coli RNAP in vitro
RNA (relative amount)*
amyP
pAMY10 1.0 1.0
pAK15 0.052 ± 0.0070 0.058 ± 0.013
pAK75 0.24 ± 0.11 0.21 ± 0.056
pAK85 0.31 ± 0.027 0.25 ± 0.016
pAK86 0.25 ± 0.047 0.17 ± 0.013
pAK19 0.98 ± 0.15 0.85 ± 0.24
pCON 1.3 ± 0.29 0.51 ± 0.082
pCON15 1.9 ± 0.37 1.1 ± 0.01
ampP2
pAMY10 1.0 1.0
pAK09 0.047 ± 0.020 0.046 ± 0.0035
*Levels of transcription were normalized to transcription from the wild-type plasmid pAMY10.

Effect of promoter mutations on transcription by E.coli RNAP

As with B.subtilis RNAP, mutations in the -16 region all reduced in vitro transcription by E.coli RNAP. A G->C transversion at position -15 caused a 94% reduction. Mutations at positions -18, -17 and -16 were transcribed at 17, 25 and 21% of the levels of amyP. The position -19 mutation, outside the -16 region, had little effect on transcription of amyP (Fig. 2b and Table 1).

Role of the -16 region in `strong' promoters

The amyP consensus promoter (amyPc) is a derivative of the amyP promoter in which the -35 and -10 regions match those of the consensus promoter. Transcription from amyPc, on plasmid pCON, was 1.3 times the level of the wild-type promoter. A second consensus promoter (amyPc15) on plasmid pCON15 contained a position -15 G->C transversion. The amyPc15 promoter was used to determine the effect of the -16 region on a promoter with consensus -35 and -10 regions. Transcription from amyPc15 was nearly twice that of the wild-type promoter and more than from amyPc (Fig. 2a and Table 1). Therefore, a promoter with consensus -35, -16 and -10 elements was stronger than the wild-type promoter, but was not the optimal sequence, as disruption of the -16 region in the consensus promoter increased utilization.

Transcription from amyPc was only 50% that of wild-type amyP when transcribed by E.coli RNAP. Transcription of the amyPc15 promoter, containing the altered -16 region, was in the range of wild-type amyP (Fig. 2b and Table 1). The consensus promoter without an intact -16 region had twice the level of transcription of the consensus promoter with the -16 region intact. The presence of three consensus promoter elements was less effective than the same promoter without the TG motif, but was worse than the wild-type promoter. Especially in the case of E.coli RNAP, it appears that the consensus promoter-RNAP interaction is too strong and may prevent efficient RNAP clearance.

Transcription from two promoters

The [alpha]-amylase gene in B.subtilis is transcribed in vivo from only one promoter, amyP, while two prominent promoters function when the [alpha]-amylase gene is expressed in E.coli (2). The secondary promoter expressed in E.coli is upstream of amyP and is designated here amyP2 (Fig. 1). This promoter contains a 17 base spacer sequence, a -35 region with a 4/6 match to the consensus -35 region, a -10 region with a 3/6 match to the consensus -10 region and initiates transcription 7 nt from the -10 region at a G. Like amyP, amyP2 contains sequences indicative of a functional -16 region with moderate matches to the consensus promoter in the -35 and -10 regions. The amyP2 -16 region sequence (TTTG) is a 3/4 match to the -16 region consensus sequence and it contains the important TG motif. While only transcripts from amyP are detectable in vivo in B.subtilis, transcripts from both amyP and amyP2 are produced in vitro with B.subtilis RNAP (Fig. 2a). Transcription by E.coli RNAP is similar in vivo and in vitro, with both promoters being functional. The relative levels of in vitro utilization between amyP and amyP2 by B.subtilis and E.coli RNAP are similar (Fig. 2). Apparently, the conditions or other factors that allow B.subtilis RNAP to be more stringent than E.coli RNAP in discriminating between promoter sequences are not present in our in vitro system. Perhaps B.subtilis RNAP requires levels of the RNAP associated proteins ([delta], [omega]1 and [omega]2) (16) or some other factor(s) that is not present in sufficient levels in our RNAP preparation to distinguish between a physiological and a non-functional promoter. Environmental conditions such as nucleotide concentrations, salt concentrations and the level of molecular crowding may also be responsible for the observed loss of selectivity in vitro. Since amyP2 contains all the elements of a functional promoter, it is possible that the presence of amyP2 is not simply chance, but performs an unknown physiological role.

amyP2 transcription

Like amyP, the amyP2 promoter is dependent upon -16 region sequences. Plasmid pAK09 contains a G->T transversion at the G in the -16 region. Just as the -15 G mutation in pAK15 eliminated transcription from amyP, the G mutation in pAK09 eliminated promoter utilization of amyP2 by both B.subtilis and E.coli RNAP (Fig. 2a and b and Table 1).

The position -15 G->C transversion in the promoters on both pAK15 and pCON15 changes the start site of amyP2 from a G to a C. The mutation shifted amyP2 initiation 1 base downstream to an A at position -14. The shift allowed for initiation at a preferred purine over the introduced pyrimidine (Fig. 2a and b). The initiating nucleotides were confirmed by in vitro transcription with [gamma]-32P-labeled NTPs. Strangely, amyP2 transcription from pCON, carrying the amyPc promoter, is altered when transcribed by E.coli RNAP. The normal size transcript from amyP2 is absent, while smaller transcripts starting at two A residues are present (Fig. 2b).

[sigma]A stimulation of transcription

During B.subtilis growth and spore formation, several [sigma] factors are produced that recognize different promoter sequences. It would be reasonable to assume that the -16 region is part of a promoter recognized by an alternative [sigma] factor, because [alpha]-amylase transcription is turned on at the end of exponential growth, a period when other [sigma] factors are active. As with many B.subtilis genes expressed during the transition and stationary phases, stimulation of [alpha]-amylase production could be a result of a secondary [sigma] factor. To demonstrate that amyP and amyP2 are transcribed by E[sigma]A, we performed in vitro transcription with E[sigma]A purified from cells harvested in exponential growth to lessen the chance of purifying RNAP containing the alternate [sigma] factors produced in stationary phase. To further demonstrate that the E[sigma]A form of RNAP would recognize both promoters and to saturate RNAP with [sigma]A, additional [sigma]A was added to the reactions. Transcription reactions were stimulated by increasing amounts of [sigma]A from 0.76 to 4.5 pmol/reaction. Transcription from both promoters was increased ~2-fold by 4.5 pmol additional [sigma]A: amyP by 110% and amyP2 by 86% (data not shown). Addition of [sigma]A to the in vitro transcription reactions increased transcription from both amyP and amyP2, confirming that these promoters can indeed be transcribed by E[sigma]A. If [sigma]A did not recognize amyP and amyP2, it would not stimulate transcription and would likely inhibit transcription, by competing with a contaminating [sigma] factor for core RNAP. Even though amyP is transcribed by E[sigma]A, it is possible that amyP can also be recognized by alternative [sigma] factors.

Analysis of B.subtilis promoter sequences

To elucidate regions other than the -16 region that may be conserved in promoters containing the -16 region, the general category of B.subtilis promoters was split into two subsets; -16 region promoters that contained the TG motif and promoters without the -16 region TG motif. Figure 3 summarizes the percentages at which the most frequent base occurs at each position for both promoter subsets. Several conserved regions were found upstream of the -35 region in the -16 region promoters, but were not found in the promoters without the TG motif. A weakly conserved An tract from positions -54 to -51, a Tn tract from positions -50 to -47 with a moderately conserved T at position -48, a moderately conserved An tract from position -44 to -40 and a CtG motif just upstream of the -35 region were all conserved in the -16 region promoters. Other conserved bases outside the -16 region were two T bases in the spacer sequence between the -35 and -10 regions and an A just downstream of the -10 region at position -6. In the -16 region (TRTG) the upstream T was found in 52% of the -16 region promoters. An A occurs in 41% of the promoters at the second position and a G in 28% of the promoters, therefore purines represent 69% of the bases at position -18.


Figure 3. Comparison of base conservation of promoters with (a) and without (b) the -16 region TG motif. Bacillus subtilis promoters from Helmann (3) with confirmed transcription start sites were divided into the 64 promoters containing the TG motif 1 base upstream of the -10 region and the 78 promoters lacking the motif. The percentage of the most frequent base is plotted at each position. Statistically significant bases are listed above the corresponding bar. Poisson statistics as described (23) were used to determine the number of standard deviations (SD) from the average occurrence of the most frequent base at each position. Bases that were found to be between 2 and 3 SD above their average occurrence are indicated by lower case letters and considered weakly conserved, while bases >3 SD above their average occurrence are indicated by upper case letters.

The promoters lacking a -16 region TG motif appear to have few conserved sequences outside the -35 and -10 regions. Four bases from -69 to -48 are weakly conserved, while the position -19 T and two A bases just downstream of the -10 region are moderately conserved.

The -35 and -10 regions were highly conserved in both sets of promoters. The -16 region promoters were more conserved at five of the six positions in the -35 region than promoters without the -16 region. The promoters without the -16 region had slightly greater conservation in the -10 region than those with a -16 region, especially at positions -12 and -11. Therefore, even though some promoters have conserved bases in the -16 region, they still have highly conserved bases in the -35 and -10 regions as well as upstream conserved sequences.

Gram-positive bacterial promoter sequences

All available compilations of gram-positive bacterial promoter sequences, with the exception of Streptomyces promoters, which do not resemble typical E[sigma]70 bacterial promoters (24), were analyzed to determine the frequency of the most common bases in the -16 region. Promoter compilations of at least 10 promoters with experimentally confirmed transcription start sites were included (Table 2). The Lactobacillus compilation is a combination of two promoter lists from a total of seven Lactobacillus species. To determine the -16 region sequences, the -10 regions were aligned and the frequency of each base 2-6 bases upstream of the -10 region was calculated. All compilations revealed significantly conserved -16 region sequences. As described (28), the TRTG motif in Streptococcus pneumoniae is highly conserved, ranging from 75 to 92%. The TRTG motif in Corynebacterium glutamicum promoters is less conserved; interestingly, the -35 and -10 regions also appear to be less conserved. It is possible that some of the promoters included in the compilations are not recognized by the major RNAP [sigma] factor analogous to [sigma]A, which would result in a compilation with lower conserved base frequency.


Table 2. Percent occurrence of bases in gram-positive bacterial promoters

DISCUSSION

Single base substitutions in the -16 region resulted in striking effects on in vitro transcription. The -15 G->C transversion nearly eliminated in vitro utilization of amyP by both B.subtilis and E.coli RNAP. Mutations at other positions in the -16 region also significantly reduced utilization of the promoter by B.subtilis and E.coli RNAP. In addition, the -16 region sequence appears to play the same vital role for the functionality of amyP2 as it does in amyP. Excess RNAP, limiting plasmid DNA and prebound DNA-RNAP complexes contributed to favorable conditions for promoter function. Even under these ideal conditions the -35 and -10 regions were insufficient for promoter function without an intact -16 region TRTG motif. The importance of the -16 region in a defined in vitro system confirms that this region is necessary for transcription by E[sigma]A and E[sigma]70. The contribution of unknown factors can be eliminated under in vitro conditions. Therefore, any factors other than RNAP which specifically recognize the -16 region are not necessary for transcription of amyP in B.subtilis and E.coli.

To better understand the importance of the -16 region in other promoters, we closely analyzed B.subtilis promoter sequences for the presence of -16 region sequences. Previous assessments of B.subtilis promoters treated all promoters as one group. In our study, promoters containing the -16 region TG motif were analyzed as a subset. Figure 3a and b demonstrates the striking differences between the -16 region subset and promoters without the -16 region. Several conserved regions upstream of the -35 region were found in the -16 region promoters that were not found in the promoters without -16 regions. Since in E.coli the `extended -10' class of promoters are missing -35 regions, we had expected that the -16 region promoters would have weakly conserved bases in the -35 region. This was not the case, as the -16 region promoters contain -35 and -10 regions which are as conserved or more so than promoters without -16 regions, as well as additional upstream conserved sequences. Therefore, it is apparent that, unlike in E.coli `extended -10 promoters', the TG motif does not replace the -35 region. In general, promoters containing the -16 region have typical -35 and -10 regions and other conserved sequences and appear to comprise a class of `strong' promoters, including most of the rRNA and many phage promoters.

Sequences from positions -54 to -40 are conserved in the -16 region promoters. The conserved sequences are An and Tn tracts similar to the UP element sequences found in E.coli rRNA promoters (30). UP element sequences function to allow docking of the C-terminal domain of the RNAP [alpha] subunit (31). There seems to be a correlation between the TG motif shared by the `extended -10 promoters' and the -16 region and the presence of UP element-like sequences. It has been reported that for `extended -10 promoters' RNAP contacts are made further upstream than in typical promoters (12). The observation of a correlation between the presence of UP element sequences and the -16 region could suggest a cooperative function between the two elements or, alternatively, they may function in an additive manner, as both elements are present in `strong' promoters with many conserved sequences. The TG motif may function independently of the UP element; not only do the [alpha]-amylase promoters lack UP element-like sequences, there is evidence that `extended -10 promoters' do not require UP element sequences for open complex formation (13,32). The absence of UP element-like sequences and a minimum of conserved bases in the -35 and -10 regions may explain why amyP and amyP2 require the -16 region.

The -16 region TRTG motif is a fourth element of bacterial promoters, along with the two primary elements of the -35 and -10 regions and the UP element. As the -16 region is not necessary for many promoters, its utility is context dependent. In promoters with adequate sequence information in other elements, like the [alpha]-amylase consensus promoter, the -16 region is not required and may even be detrimental for promoter function. In the `extended -10' class of promoters, which lack conserved -35 regions, and in some promoters with identifiable -35 regions, such as wild-type amyP, the -16 region is essential. An explanation of the function of the -16 region may involve the ability of the motif to enhance a step in transcription initiation which is typically performed by the -35 region. In `extended -10 promoters' the TG motif is known to compensate for the lack of a -35 region (33), while in promoters with a -35 region the -16 region may function in an additive manner to promote or stabilize a rate limiting initiation step. One model of the steps in open complex formation involves the spacer DNA being untwisted by RNAP to align the -35 and -10 regions on the same face of the double helix and interact with RNAP. The untwisting of the spacer DNA places stress on the DNA, resulting in melting of the double helix at the -10 region (34). By this model, spacer DNA sequences that could contribute to the untwisting or bending of the spacer would enhance the rate of formation or stability of the initial open complex. Furthermore, at `extended -10 promoters' RNAP would not be anchored at the -35 region and, as a consequence, would be unable to untwist or bend the spacer DNA without compensation by interactions and bending in the -16 region.

Compilations of gram-positive bacterial promoters are few; even so, it is evident that the TRTG motif is widespread. The motif is rare in gram-negative bacterial promoters, however, it is vital for transcription by E.coli RNAP from `extended -10 promoters' and from the B.subtilis [alpha]-amylase promoters when placed in E.coli. In Campylobacter jejuni, a member of a gram-negative group distantly related to other eubacteria (35), a -16 region-like sequence is highly conserved (36). The sequence TTTTTTTG is in the same relative position to the -10 region as is the -16 region. Campylobacter jejuni promoters have -10 regions similar to promoters recognized by B.subtilis E[sigma]A and E.coli E[sigma]70, but have conserved -35 regions that share little similarity with their B.subtilis and E.coli promoter counterparts (36). It has been hypothesized that the TG motif along with the -10 region was an early promoter configuration from which contemporary promoters evolved, as it is the minimum sequence necessary for promoter function in E.coli (37). If so, then E.coli and other gram-negative bacteria that have evolved along a similar path as E.coli have for the most part lost the TG promoter motif, while many gram-positive bacteria, along with some other lineages of eubacteria, have retained it. In a related study, Solnick et al. (38) found the rate of amino acid change in RpoD of gram-negative bacteria to be three times faster than in gram-positive bacteria. This would imply that gram-positive bacterial RNAPs have diverged less than their gram-negative counterparts from a eubacterial ancestral RNAP and correlates well with the observation that RNAPs from gram-positive bacteria require promoter sequences that may represent an earlier promoter form than do gram-negative bacterial RNAPs. Gram-positive bacterial RNAPs appear to commonly utilize several elements in addition to the -35 and -10 regions, although none of the additional elements appears to be essential for all promoters. More likely, different combinations of these elements can form functional promoters.

ACKNOWLEDGEMENTS

We thank Dr John D.Helmann for providing easy access to his compilation of B.subtilis promoters on the World Wide Web. We also thank Richard L.Gourse and M.J.Rosovitz for discussions and help with the manuscript and Dr Bang-Yang Chang and Dr Roy H.Doi for E.coli strain BL21/DE3 containing plasmid pCD2, used for overproduction of [sigma]A. This work was supported in part by the College of Agriculture, University of Wisconsin, National Institutes of Health grant GM34324 and National Institutes of Health Service Research Award T32GM08349.

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*To whom correspondence should be addressed. Tel. +1 608 263 2914; Fax. +1 608 262 9865; Email: ghchambl@facstaff.wisc.edu

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