Mutational analysis of the promoter recognized by Chlamydia and Escherichia coli sigma(28) RNA polymerase

Abstract

sigma(28) RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial sigma(28) promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the sigma(28)-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a -35 element recognized by chlamydial sigma(28) RNA polymerase that resembles the consensus -35 sequence. Within the -10 element, however, chlamydial sigma(28) RNA polymerase showed a striking preference for a CGA sequence at positions -12 to -10 rather than the longer consensus -10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli sigma(28) RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the -10 promoter element recognized by sigma(28) RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that sigma(28) RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for sigma(28) RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred sigma(28) promoter that we defined in the context of the hctB promoter is TAAAGwwy-n(11/12)-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n(11/12) is a spacer of 11 or 12 nt.

FIG. 1.

The sequence of the hctB promoter aligned with bacterial core and extended σ²⁸ consensus sequences. The −35 and −10 promoter elements are separated by a spacer length of 11 or 12 nt.

FIG. 2.

Effect of point substitutions within the hctB promoter on in vitro transcription by C. trachomatis σ²⁸ RNA polymerase. All three possible point substitutions were tested at each position in the −35 element from −37 to −24 (A) and in the −10 element from −17 to −4 (C). The wild-type sequence of the predicted element is shown below each graph. Changes in promoter activity are depicted as the decrease (n-fold) relative to wild-type promoter activity. Decreases greater than 200-fold are not shown as extending below the bottom axis. Each bar represents the mean of three independent experiments. Sample transcription of DNA templates containing the wild-type (wt) hctB promoter and point substitutions of positions −31 (B) and −11 (D) are shown.

FIG. 3.

Effect of the spacer length on transcription. (A) In vitro transcription by C. trachomatis σ²⁸ RNA polymerase of hctB promoter templates containing a spacer of 9 to 13 nt as indicated. The 12-nt wild-type (wt) spacer is marked for reference. (B) Graph showing quantification of the transcription results for C. trachomatis and E. coli RNA polymerases. Reactions were performed in triplicate, and standard deviations are marked by error bars. Results for each RNA polymerase were normalized to a promoter activity of 100% for the wild-type hctB promoter.

FIG. 4.

Effect of point substitutions within the hctB promoter on in vitro transcription by E. coli σ²⁸ RNA polymerase. All three possible point substitutions were tested at each position from −37 to −24 (A) and from −17 to −4 (B). The wild-type sequence of each predicted promoter element is shown below the respective graph. Changes in promoter activity are shown as the decrease (n-fold) relative to wild-type promoter activity. Decreases greater than 200-fold are not shown as extending below the bottom axis. Each bar represents the mean of three independent experiments.

FIG. 5.

Sequence logos for the −35 and −10 elements of the σ²⁸-dependent promoter. (A) Sequence recognized by C. trachomatis σ²⁸ RNA polymerase in the context of the C. trachomatis hctB promoter. (B) Sequence recognized by E. coli σ²⁸ RNA polymerase in the same promoter context. (C) The sequence logo based on the nucleotide frequencies of known bacterial σ²⁸ promoters. Details of the sequence logo format are presented in the Materials and Methods and Results sections. All sequence logos were derived using WebLogo, which is available online at http://weblogo.berkeley.edu.