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

Department of Microbiology and Molecular Genetics,¹ Department of Medicine, School of Medicine, University of California-Irvine, Irvine, California 92697-4025²

Received 5 April 2006/ Accepted 15 May 2006

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

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