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

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.     

Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella

Flagellar class 3 operons of Escherichia coli and Salmonella are transcribed by RNA polymerase containing sigma 28. The consensus sequence of the sigma 28-dependent promoters was believed to be TAAA N15 GCCGATAA. In this study, we found that the E. coli genome contains a large number of sequences homologous to this consensus. However, we showed that they do not always exert a sigma 28-dependent promoter activity. We compare more carefully the sequences of the class 3 flagellar promoters and propose a revised structure of the sigma 28-dependent promoters as TAAAGTTT N11 GCCGATAA.     

Functional interaction between RNA polymerase alpha subunit C-terminal domain and sigma70 in UP-element- and activator-dependent transcription

We show that the Escherichia coli RNA polymerase (RNAP) alpha subunit C-terminal domain (alphaCTD) functionally interacts with sigma(70) at a subset of UP-element- and activator-dependent promoters, we define the determinants of alphaCTD and sigma(70) required for the interaction, and we present a structural model for the interaction. The alphaCTD-sigma(70) interaction spans the upstream promoter and core promoter, thereby linking recognition of UP-elements and activators in the upstream promoter with recognition of the -35 element in the core promoter. We propose that the alphaCTD-sigma(70) interaction permits UP-elements and activators not only to "recruit" RNAP through direct interaction with alphaCTD, but also to "remodel" RNAP-core-promoter interaction through indirect, alphaCTD-bridged interactions with sigma(70).