INTRODUCTION

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Chlamydia trachomatis is a gram-negative obligate intracellular pathogen with a biphasic developmental life cycle (reviewed in references 16 and 20). We have been characterizing the transcriptional machinery and defining the structure of chlamydial promoters. C. trachomatis RNA polymerase (RNAP) resembles other eubacterial RNAPs in being a multisubunit enzyme containing , , ', and  subunits (3, 4, 11, 12). Three  subunits have been identified in Chlamydia to date. ^A is a homolog of ⁷⁰, and two alternative  subunits show sequence homology to Escherichia coli ⁵⁴ and Bacillus subtilis ²⁸, respectively (21). ^A shows striking amino acid sequence conservation with ⁷⁰ in subregions 2.4 and 4.2 (3, 12), which recognize the 10 and 35 promoter elements, respectively. Within these regions, specific amino acid residues that have been shown to be involved in promoter recognition are completely conserved (14).

It is not clear if this striking conservation in the  promoter recognition domains is accompanied by conservation of promoter structure between C. trachomatis and E. coli. Consensus ⁷⁰ 10 promoter sequences can be recognized upstream of the transcription initiation sites of many C. trachomatis genes (7). Some, but not all, of these genes also have 35 promoter sequences spaced an optimal 16 to 18 bp upstream of the 10 promoter sequences (6, 8, 13, 23). However, other chlamydial genes are not preceded by recognizable ⁷⁰ promoter sequences.

In vitro transcription studies have begun to define the structures of promoters in C. trachomatis. Matthews and Sriprakash found that in vitro promoter activity was reduced or eliminated by multiple mutations in the predicted 10 and 35 regions of the plasmid countertranscript (PCT) promoter, although single substitutions had no measurable effect (15). Douglas and Hatch showed that sequences in the 35 region and adjacent AT-rich regions were important for major outer membrane protein (MOMP) P2 promoter activity in vitro (2). The predicted 10 hexamer of MOMP P2, TATCGC, differed from the E. coli 10 hexamer by the presence of C and G residues in the last three positions, but only the first two bases of this hexamer were important for promoter activity.

We have been characterizing C. trachomatis rRNA P1 by in vitro transcription with heparin-agarose-purified C. trachomatis RNAP. rRNA is highly transcribed, and rRNA P1 is likely to be a strong promoter. In E. coli, the rRNA promoters are among the strongest promoters and are very close in sequence to the consensus ⁷⁰ promoter. E. coli rRNA promoters also contain a third promoter element, the UP element, that can enhance transcription as much as 30-fold (17). Unlike 10 and 35 promoter elements, which are recognized by ⁷⁰, the UP element is recognized by the carboxy-terminal domain of the  subunit of RNAP (-CTD). While UP elements have not been previously identified in C. trachomatis, the amino acids of that recognize the UP element are completely conserved between E. coli and C. trachomatis (10, 11).

In our previous study, we used 5' deletions and 5-bp substitutions of rRNA P1 to demonstrate that sequences in the approximate 10 and 35 regions were required for promoter activity with C. trachomatis or E. coli ⁷⁰ RNAP (22). This analysis also defined a region within 26 to 22 that was required for transcription by partially purified C. trachomatis RNAP but not by E. coli RNAP.

To further define the specific bases required for transcription by C. trachomatis RNAP, we performed saturation mutagenesis of rRNA P1 by constructing single base substitutions from 41 to 1. The effects of these substitutions on transcription by C. trachomatis and E. coli RNAPs were compared. Our results defined four regions that contributed to in vitro transcription by partially purified C. trachomatis RNAP.

	MATERIALS AND METHODS

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Reagents. Products were obtained from the following sources and were used according to the manufacturers' specifications. Restriction enzymes, bacterial alkaline phosphatase, and T4 DNA ligase were from Gibco BRL (Gaithersburg, Md.); T4 polynucleotide kinase was from Boehringer Mannheim Biochemicals (Indianapolis, Ind.); RNasin and RQ1 DNase were from Promega Biotech (Madison, Wis.); SP6 RNA polymerase was from Ambion (Austin, Tex.); Sequenase DNA polymerase was from United States Biochemical Corp. (Cleveland, Ohio); Thermus aquaticus DNA polymerase was from Cetus Corp. (Emeryville, Calif.); ³²P-containing nucleoside triphosphates were from Amersham Corp. (Arlington Heights, Ill.); SeaPlaque and SeaKem agarose were from FMC Bioproducts (Rockland, Maine); ampicillin and gentamicin sulfate were from Sigma Chemical Co. (St. Louis, Mo.); vancomycin hydrochloride was from Abbott Laboratories (North Chicago, Ill.), and dimethyl sulfoxide was from Fisher Scientific (Pittsburgh, Pa.).

DNA manipulation. Standard recombinant DNA methods were used for nucleic acid preparation and analysis (18). DNA was amplified by PCR as described previously (22). DNA sequencing was performed with the dideoxy-chain termination method (19) using a Sequenase kit (United States Biochemical Corp.) on double-stranded plasmid DNA.

Synthetic oligonucleotides. The following single-stranded oligonucleotide primers were synthesized by Gibco BRL: M13 forward 40, 5' GTTTT CCCAG TCACG AC; M13 reverse 40, 5' GTTGT GTGGA ATTGT G; pGLS3', 5' ATAGG AGGAA TAATG; rRNA-3, 5' CAGGG TACCA GGCCT CCGCG TTCAA GA; rRNA-4, 5' CACGA ATTCC GCGTT CAAGA AAGG; Tx1, 5' AAAGT AACAT CTTAT ATCAA CCTCT; Tx25, 5' TATTA TATAG TGTCA CCTAA AT; Tx27, 5' GGGAA GAGGG GGTGA GAG; and Tx33, 5' CCGAA CGACC GAGCG CAGCG.

Plasmid containing the rRNA P1 and SP6 control promoters. pMT589 (Fig. 1) was derived from pMT504 (22) by deletion of the lacZ and T7 promoters on a 124-bp PvuII-ApaI fragment and construction of a new SP6 control transcription template. The SP6 promoter was amplified by PCR, using pGEM-7Zf(+) as the template with M13 reverse 40 and Tx25 primers, and cloned upstream of the control G-less cassette. The G residue at +1 was replaced with a T residue to allow for transcription in the absence of GTP. Transcription with SP6 RNAP produced a 130-nucleotide control transcript.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Transcription template plasmid with rRNA P1 test promoter and SP6 control promoter. Wild-type rRNA P1 (302 to +5), or promoter templates containing mutations, was cloned immediately upstream of the test G-less cassette in plasmid pMT589. In vitro transcription with C. trachomatis RNAP or E. coli RNAP produced a 158-nucleotide (nt) test transcript. The control promoter region contained an SP6 promoter cloned in front of the control G-less cassette. Transcription with SP6 RNAP produced a 130-nucleotide control transcript.

Construction of rRNA P1 templates containing mutations. A 300-bp region of rRNA P1 (302 to +5) was generated by PCR or by the megaprimer method (see below). PCR was used if the desired mutation was located close to the transcription start site so that it could be introduced on a PCR primer. The promoter region was amplified by using pMT513 (22) as the template, rRNA-4 as the 5' primer, and a 3' primer containing the mutation. The 300-bp PCR product was phosphorylated, digested with EcoRI, and ligated to plasmid pMT589 previously digested with EcoRI and EcoRV.

Megaprimer method for generating promoter templates with mutations. A modification of the megaprimer method (22) was used to introduce mutations that could not be located on a primer for one-step PCR amplification. The megaprimer was generated by PCR with pMT513 as the template, pGLS3' as the 3' primer, and a mutation-containing 5' primer. The 200-bp megaprimer was electrophoresed and recovered from an agarose gel (22). The megaprimer was extended with Sequenase DNA polymerase, using 100 ng of heat-denatured, linearized plasmid pMT175 (22) as the template in a 10-µl reaction volume. Two microliters of the extended megaprimer mix was used as the template in a second PCR with 100 pmol of primers Tx33 and Tx27 and 10% (vol/vol) dimethyl sulfoxide. The 600-bp PCR product was recovered from an agarose gel, digested with EcoRI and DraI, and ligated to plasmid pMT589 previously digested with EcoRI and EcoRV.

In vitro transcription. The method used is as described in reference 22 with slight modifications. The following reaction mixture was assembled and preincubated on ice for 30 min: 50 mM potassium acetate, 8.1 mM magnesium acetate, 50 mM Tris acetate (pH 8.0), 27 mM ammonium acetate, 2 mM dithiothreitol, 400 µM ATP, 400 µM UTP, 1.2 µM CTP, 0.21 µM [-³²P]CTP (3,000 Ci/mmol), 100 µM 3'-O-methylguanosine 5'-triphosphate, Na salt (Pharmacia Biotech, Piscataway, N.J.), 18 U of RNasin, 10% glycerol, and 5 U of SP6 RNA polymerase. The DNA template (final concentration, 25 nM) and 0.25 µl of heparin-agarose-purified C. trachomatis RNAP (22) were added, and the reaction mixture was incubated at 37°C for 5 min. Heparin was added to a final concentration of 150 µg/ml, and the incubation was continued at 37°C for a further 10 min. The final reaction volume was 10 µl. The reaction was stopped by the addition of 10 µl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol); 7 µl of the sample was electrophoresed on an 8 M urea-6% polyacrylamide gel. Transcripts were visualized by autoradiography and quantified with a Molecular Dynamics (Sunnyvale, Calif.) PhosphorImager. The size of the transcript was determined by coelectrophoresis with an M13 sequencing ladder. A single preparation of C. trachomatis RNAP was used for the transcription reactions in this study.

Calculation of promoter activity. For each promoter template, transcription with C. trachomatis RNAP (or E. coli RNAP) was normalized to a control SP6 RNAP transcript, which corrected for DNA concentration and sample handling. The promoter activity obtained with wild-type rRNA P1 was defined as 100%, and the promoter activity of each mutant promoter was normalized to this value. For each mutant promoter, three measurements of promoter activity were obtained and a mean and standard deviation were calculated.

Primer extension analysis of in vitro transcription products. Unlabeled in vitro transcripts were synthesized as described above, with the following changes: each 10-µl reaction mixture contained 2 µl of C. trachomatis RNAP (or 0.05 U of E. coli RNAP), 400 µM CTP, and no [-³²P]CTP. Prior to primer extension analysis, RNA samples were treated with RQ1 DNase to remove the DNA template. Primer extension analysis was carried out as previously described (5) with ³²P-end-labeled primer Tx27, which is complementary to the extreme 3'-end of the test G-less cassette template. Primer extension products were electrophoresed next to a Tx27-primed DNA sequence of pMT513 (22).

	RESULTS

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We have renumbered the positions of rRNA P1 since our previous study (22). We have shifted +1 downstream one position, as repeat primer extension studies have localized the in vitro transcription initiation site to this position (data not shown). The base at the new +1 position is a G residue. The three adjacent in vivo start sites (5) are now at positions 1, +1, and +2. The inefficiency of the C. trachomatis transcription system required that we employ a G-less transcript sequence and use transcription reactions in which no GTP was added in order to increase sensitivity and permit quantitation of in vitro transcription (22). To allow transcription in the absence of GTP, we have made a substitution of G to A at +1. We cannot rule out the possibility that this substitution altered the properties of the promoter. Nevertheless, since E. coli RNAP does not have a preference for G over A at +1 so long as the initiating nucleoside triphosphate is provided at a high concentration (reference 9 and unpublished results), we have made the assumption that C. trachomatis RNAP recognizes rRNA P1 promoters similarly with A or G at this position.

We tested the effect on in vitro transcription of single base substitutions at positions 41 to 1 of rRNA P1 (Table 1 and Fig. 2). All three substitutions were tested for 37 of 41 positions, and two substitutions were tested for each of the remaining positions. Mutations with effects on transcription clustered in several regions that were specific for C. trachomatis and E. coli RNAPs.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Effects of single substitutions on C. trachomatis rRNA P1 transcription by E. coli or C. trachomatis RNAPs. All three possible substitutions were tested from 41 to 1, except 41, 19, 18 and 16, where only two were tested (Table 1). The wild-type rRNA P1 sequence is shown on each graph, and changes in promoter activity resulting from the substitutions, relative to the activity of the wild-type promoter, are indicated. Decreases larger than fivefold are not illustrated as extending beyond the bottom axis.

Substitutions in the 10 region affected promoter activity. Substitutions in the 10 region had large effects on transcription by C. trachomatis RNAP and identified positions that were important for promoter activity. Some substitutions at 11, 10, 9, 8, 7, 5, and 4 had negative effects on promoter activity, while other substitutions at 10 and 6 had positive effects (Table 1 and Fig. 2). The greatest effects were seen at 8, where an A-to-G substitution caused a ninefold decrease in promoter activity, and at 9, where a T-to-A substitution decreased promoter activity fivefold. The positive effects of a G substitution at 10 and a T substitution at 6 suggested that these substitutions made the resultant promoter closer to the optimal promoter sequence. If we took the individual base(s) that produced the greatest promoter activity at each position, the sequence was A, G, or T at 11, G or T at 10, T at 9, A at 8, A or T at 7, A or T at 6, A or C at 5, and T at 4. This optimal sequence contains two potential matches to the ⁷⁰ 10 promoter element, TATAAT, from 11 to 6 and from 9 to 4.

Substitutions in the 35 region had smaller effects on C. trachomatis RNAP than substitutions in the 10 region. We have previously shown that a 5-bp substitution from 31 to 27 decreased transcription by C. trachomatis RNAP (22). Single substitutions at 32 and 31 had negative effects, while substitutions 33, 29, and 28 had positive effects (Table 1 and Fig. 2). Substitutions at 30 had a slight positive effect. The largest effect was a T-to-C or G substitution at 32 which decreased promoter activity twofold. The optimal sequence was T at 33, T at 32, A, G or T at 31, G at 29, and A at 28, which resembles the ⁷⁰ 35 promoter element, TTGACA. The optimal 35 sequence for E. coli RNAP was T at 33, T at 32, A, G or T at 31, G at 30, A at 29, A at 28, and A at 27 (Table 1 and Fig. 2). Two overlapping potential 35 elements that resemble the E. coli consensus 35 hexamer are recognizable, from 33 to 28 and 32 to 27.

Substitutions between the 10 and 35 regions defined an AT-rich region that affected transcription by C. trachomatis RNAP but not by E. coli RNAP. Our previous analysis suggested that sequences in the spacer region between 10 and 35 regions were important for transcription by C. trachomatis RNAP but not by E. coli RNAP (22). At positions 26 to 22, changing the wild-type sequence from AAAAA to TTTTT caused a 2-fold decrease in promoter activity, and altering it to CCCCC resulted in a 12-fold decrease in promoter activity (data not shown). Within this region, substitution of a single wild-type A residue with a C or G at 25, 24, and 23 or a C at 22 caused a negative effect on transcription (Table 1 and Fig. 2 and 3). Substitution of a C at 26 had a smaller negative effect. Substitution of a T residue at 26, 25, and 23 had a slight stimulatory effect. The greatest effect was seen at 24, where an A-to-C substitution caused a 4.5-fold decrease in promoter activity. In contrast, transcription by E. coli RNAP was slightly decreased by C or G substitutions at 23, while other substitutions in this region had no effect or a slight positive effect (Table 1 and Fig. 2 and 3). These results defined a region extending from 25 to 22 and possibly including 26, whose sequence was important for transcription by C. trachomatis RNAP. We have called this region the spacer A/T region because of its location in the spacer region between the 10 and 35 promoter elements and the sequence preference for A and T residues.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   In vitro transcription of C. trachomatis rRNA P1 templates with C. trachomatis RNAP (A) or E. coli RNAP (B). The rRNA P1 templates contained the wild-type (wt) sequence (lane 1), a substitution of wild-type A at 23 with a C (lane 2), G (lane 3), or T (lane 4), or a substitution of wild-type A at 24 with a C (lane 5), G (lane 6), or T (lane 7). The shorter control transcript was transcribed by SP6 RNAP.

Substitutions upstream of the 35 region affected transcription. We have previously shown that 5-bp substitutions from 41 to 37 and 36 to 32 decreased transcription by C. trachomatis and E. coli RNAPs (22). Some single base substitutions in this region upstream of the putative 35 hexamer affected transcription by C. trachomatis RNAP, although not as much as they affected transcription by E. coli RNAP (Table 1 and Fig. 2). Most of these deleterious substitutions were from an A to a C or G. As we will mention below, the native AT-rich sequence in this location functioned as an UP element with E. coli RNAP. Thus, it is likely that the substitutions decreased promoter activity by affecting contacts between the UP element and RNAP.

Effect of altered spacing between the 10, spacer A/T, and 35 regions. We tested the effect on promoter activity of insertions or deletions downstream (at 17) or upstream (at 27) of the spacer A/T region to determine if the spacing between the 10, spacer A/T, and 35 regions was important (Fig. 4). A 1-bp insertion or deletion at either location had minimal effects on transcription by C. trachomatis RNAP (lines B, C, F, and G). A 2-bp insertion at 27 (line E) but not 17 (line D) had a negative effect on C. trachomatis and E. coli RNAPs. The only mutation which specifically decreased transcription by C. trachomatis RNAP but not E. coli RNAP was a 2-bp deletion at 17 (line H).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Spacing mutations of C. trachomatis rRNA P1 and their effects on transcription by C. trachomatis RNAP and E. coli RNAP. The names of the spacing mutations are abbreviated so that +1 @ 17 represents a 1-bp insertion at position 17. Inserted sequences are shown in uppercase letters, and the site of deletion is marked with a . The sequences are aligned by the transcription initiation site. The G residue at +1 in the native sequence was replaced by an A residue to allow for transcription in the absence of GTP. Positions that were shown by the substitution analysis to be important for C. trachomatis RNAP promoter activity are underlined in the wild-type sequence and in their new locations in the spacing mutations. The spacer A/T region is underlined twice. The promoter activity of each spacing mutation is shown for transcription by C. trachomatis RNAP (Ct) and E. coli RNAP (Ec).

Substitutions that allowed transcription initiation from a new site. Three single base changes each produced an additional C. trachomatis RNAP transcript that was faster-migrating and less abundant (Fig. 5A). The 5' end of each extra transcript was mapped by primer extension to a site 18 downstream of the native rRNA P1 transcription start site (data not shown). These results demonstrated that the extra transcript was the result of transcription initiation at a new site, rather than premature termination. Each of the substitutions appeared to make new promoters by creating sequences that could function as 35 hexamers (T-11C) or spacer A/T regions (G-6A and G-6T) in combination with a potential 10 hexamer (TTTAAA) from the G-less cassette (Fig. 5B). E. coli RNAP only produced an extra transcript with the substitution that created a perfect 35 hexamer (T-11C), providing further evidence that the effect of the spacer A/T region is specific to C. trachomatis RNAP. These results suggest that a 35 hexamer, with the same sequence as the E. coli consensus 35 promoter element (TTGACA), and the spacer A/T region can serve as important elements for recognition by C. trachomatis RNAP.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   (A) In vitro transcription of wild-type rRNA P1 and substitutions that produced an extra transcript. Lanes 1 and 5, wild-type (wt) rRNA P1; lanes 2 and 6, G-to-A substitution at 6 (G-6A); lanes 3 and 7, G-to-T substitution at 6 (G-6T); lanes 4 and 8, T-to-C substitution at 11 (T-11C). The test transcript was transcribed by C. trachomatis (Ct) RNAP (lanes 1 to 4) or E. coli (Ec) RNAP (lanes 5 to 8). The extra transcript in lanes 2, 3, 4, and 8 (marked by an asterisk) was shown to initiate 18 nucleotides downstream by primer extension (data not shown). The control transcript was transcribed by SP6 RNAP. (B) Sequences upstream of the initiation sites for the rRNA P1 transcripts shown in panel A. The sequences were aligned by the transcription initiation sites. The wild-type rRNA P1 sequence is shown in the top line, and the positions where C. trachomatis RNAP transcription was affected by substitutions are underlined. The spacer A/T region is underlined twice, and the location of a 12-bp direct repeat sequence is shown by a pair of arrows. The substituted base is shaded in each of the three mutant promoter templates. Putative 35 and 10 hexamers are underlined for T-11C, which was transcribed by both E. coli and C. trachomatis RNAPs. Proposed spacer A/T regions in G-6A and G-6T are underlined twice.

Transcription of the C. trachomatis rRNA P1 promoter was affected by deletion of the -CTD of E. coli RNAP. Our previous results demonstrated that the removal of AT-rich sequences upstream of the putative 35 region significantly decreased transcription by both C. trachomatis and E. coli RNAP (22). Enhancement of transcription by an AT-rich region upstream of an rRNA gene is suggestive of an UP element (17). To test whether E. coli RNAP could recognize this C. trachomatis sequence as an UP element, we compared transcription by wild-type E. coli RNAP and mutant E. coli RNAP that was unable to recognize the UP element because of deletion of the -CTD. With wild type E. coli RNAP, transcription of a promoter template containing the proposed UP element (302 to +5 of C. trachomatis rRNA P1) was 11-fold higher than transcription of a promoter template lacking the UP element (34 to +5) (Fig. 6, lanes 1 and 2). The stimulation of transcription was lost when mutant E. coli RNAP lacking the -CTD was used (lanes 4 and 5). These results show that C. trachomatis rRNA P1 contains an UP element that is recognized by E. coli RNAP. Since the residues in  responsible for UP element recognition are conserved between E. coli and C. trachomatis (10, 11), these data suggest that C. trachomatis RNAP also utilizes the rRNA P1 UP element.

View larger version (35K): [in this window] [in a new window]   FIG. 6.   In vitro transcription using wild-type E. coli RNAP () or E. coli RNAP with a deletion of the -CTD (-del 235). C. trachomatis rRNA P1 templates contained an UP element (wild type [wt]; 302 to +5) or lacked an UP element (34; 34 to +5). For the 34 template, the sequence that replaced the UP element was derived from the plasmid (sequence in 34 from 64 to 35: GTCGCATGCTCCTCTAGACTCGAGGAATTC. The E. coli lacUV5 promoter, which lacks an UP element (17), was used to normalize the activities of the two RNAP preparations.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have identified four regions of C. trachomatis rRNA P1 that contributed to the promoter activity of C. trachomatis RNAP: a 10 region, a 35 region, a novel spacer A/T region just downstream of the 35 region, and an UP element. Three of these regions are analogous to the corresponding regions in E. coli promoters, but the spacer A/T region was recognized only by C. trachomatis RNAP.

Context effects limit the conclusions that can be made from the single base substitutions. This is apparent in the analyses of the effect of substitutions in the 35 region. Apparently, C. trachomatis RNAP does not require a perfect 35 hexamer in the context of the wild-type rRNA P1 promoter sequence. However, in the T-11C mutant (Fig. 5B), a 35 hexamer identical to the E. coli consensus directs C. trachomatis RNAP to recognize a new promoter even though there is an imperfect spacer A/T region. On the other hand, creation of a good spacer A/T region is sufficient to allow for transcription when the 35 sequence is imperfect (G-6A and G-6T [Fig. 5B]). It is tempting to speculate that the 35 region and the spacer A/T region work cooperatively to anchor C. trachomatis RNAP to the promoter.

The promoter sequences identified for C. trachomatis RNAP resemble the E. coli ⁷⁰ promoter structure. This is consistent with the conservation of amino acid sequence between ^A and ⁷⁰ in subregions 2.4 and 4.2 (3, 12). Most substitutions in the 10 and 35 regions of rRNA P1 that affected transcription by C. trachomatis RNAP also affected transcription by E. coli RNAP, although there were subtle differences in the recognition properties of the two enzymes. For example, C. trachomatis RNAP did not recognize the extended 10 motif in this promoter context. However, subregion 2.5 of ⁷⁰, which is the region that recognizes this motif, is well conserved between ^A and ⁷⁰ (1). The particular amino acid (E458) proposed to recognize the G residue of the TG motif is also conserved.

Our most striking result is the delineation of the spacer A/T region, which was important for transcription by C. trachomatis RNAP but not E. coli RNAP. Substitutions in this region demonstrated an AT sequence preference, and the spacing mutations showed that the location of the spacer A/T region was also important.

The role of the spacer A/T region may be explained in several ways. If the spacer A/T region can function as an additional promoter element, it may be required for specific contacts with C. trachomatis RNAP. In fact, the spacer A/T region is immediately downstream of the predicted location of the 35 promoter element, and the two regions might form an extended 35 promoter element. Alternatively, the spacer A/T region could serve as a binding site for a putative transcription factor present in the C. trachomatis RNAP preparation. A third possibility is that the preference for A or T residues in this region may reflect the energetic contribution of these sequences to promoter activity, such as with localized DNA strand melting. Finally, the spacer A/T region is part of six consecutive A residues, which could produce a nonstandard DNA structure that may affect promoter recognition by C. trachomatis RNAP.

We propose that an AT-rich region from 52 to 34 functions as an UP element to increase transcription from C. trachomatis rRNA P1. We demonstrated a fourfold increase in transcription with C. trachomatis RNAP (reference 22 and data not shown) and an 11-fold enhancement with E. coli RNAP (reference 22 and Fig. 6). The effect of the UP element on E. coli RNAP transcription was dependent on the -CTD, strongly suggesting that the E. coli RNAP  subunit can recognize this heterologous UP element. We note that it is not yet technically possible to confirm that the C. trachomatis RNAP -CTD makes a contact with the UP element, as this experiment will require the construction of mutant C. trachomatis RNAP lacking the -CTD.

Interestingly, the UP element included a 12-bp sequence from 44 to 33 which was directly repeated from 29 to 18 (Fig. 5B). The downstream repeat encompasses the spacer A/T region. As the  subunit of RNAP recognizes the sequence of the UP element, it is intriguing to speculate if the  subunit can also recognize the spacer A/T region.

Our results are consistent with the analysis of C. trachomatis MOMP P2 by Douglas and Hatch (2). They reported that the first two positions of the putative 10 hexamer were important for promoter activity, and in our analysis, the first two positions of the potential 10 hexamer from 9 to 4 also showed the greatest effect with substitution; 2-bp substitutions in the AT-rich sequences upstream and downstream of the predicted MOMP P2 35 promoter element greatly abrogated promoter activity. We propose that the AT-rich sequence upstream of the 35 promoter element of MOMP P2 is an UP element, which would explain the decreased promoter activity with deletion of sequences upstream of 39 or with a 2-bp substitution at 41 and 40. We also suggest that the AT-rich region downstream of the 35 promoter element is the equivalent of a spacer A/T region. Loss of promoter activity with a 2-bp substitution at 29 and 28 (equivalent to 26 and 25 of rRNA P1 [Fig. 7]) can be explained by alteration of the spacer A/T region from AAAA to GAAA.

View larger version (43K): [in this window] [in a new window]   FIG. 7.   Alignment of selected chlamydial promoters. Upstream sequences of chlamydial genes with 70-like 10 hexamers were aligned by these putative 10 elements. Predicted 35 and 10 promoter sequences are underlined. Potential spacer A/T regions are underlined twice. In vivo transcription initiation sites are marked with a dot above the sequence. Upstream sequences of the following genes from C. trachomatis serovars L1, L2, and MoPn (mouse pneumonitis) and C. psittaci 6BC are shown (GenBank accession numbers in parentheses): L1 60-kDa CRP P1 and 15-kDa SRP (M35148), L2 MOMP P2 (M14738), MoPn S1 (M23000), L2 PCT (plasmid countertranscript) and prom7 (X07547), 6BC EUO (L13598), L2 hctA (M60902), L2 ltuA (L40822), L2 ltuB (L40838), MoPn groE (L12004), MoPn dnaK (M62819), and L2 hctB (L10193).

Analysis of other chlamydial promoters shows that many of them contain potential ⁷⁰ 10 and 35 promoter sequences (Fig. 7). A potential spacer A/T region can be identified immediately downstream of the putative 35 element in many of these promoters. The potential spacer A/T region is often part of a larger AT-rich sequence (6-8 bp). A notable exception is the dnaK promoter, which lacks an identifiable spacer A/T region but has a 5-of-6-bp match to the E. coli consensus 35 hexamer and a perfect E. coli 10 hexamer (23). This ⁷⁰-like promoter structure may be sufficient for transcription by C. trachomatis RNAP. However, for other C. trachomatis promoters, including rRNA P1, the spacer A/T region may compensate or substitute for a suboptimal 35 element.

At this time, we cannot distinguish between the likely roles of the spacer A/T region as a promoter element that interacts differently with C. trachomatis and E. coli RNAPs or as a binding site for an activator of transcription that is present in the C. trachomatis RNAP preparation. Further characterization of the spacer A/T region will require purification of C. trachomatis RNAP to homogeneity.