Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ 28 RNA Polymerase

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Abstract
σ 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 σ 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 σ 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 σ 28 RNA polymerase that resembles the consensus −35 sequence. Within the −10 element, however, chlamydial σ 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 σ 28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the −10 promoter element recognized by σ 28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that σ 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 σ 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 σ 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.