Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro

Abstract

We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential -10 and -35 elements, analogous to Escherichia coli promoters recognized by E-sigma70. We identified a novel AT-rich region immediately downstream of the -35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the -35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

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.

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.

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.

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 Δ. 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).

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.

FIG. 6

In vitro transcription using wild-type E. coli RNAP (α) or E. coli RNAP with a deletion of the α-CTD (α-del 235). C. trachomatis rRNA P1 templates contained an UP element (wild type [wt]; −302 to +5) or lacked an UP element (Δ34; −34 to +5). For the Δ34 template, the sequence that replaced the UP element was derived from the plasmid (sequence in Δ34 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.

FIG. 7

Alignment of selected chlamydial promoters. Upstream sequences of chlamydial genes with ς⁷⁰-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).