Distinct functions of regions 1.1 and 1.2 of RNA polymerase σ subunits from Escherichia coli and Thermus aquaticus in transcription initiation

RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ(70) and T. aquaticus σ(A) subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ(70) subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the -10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.     

Role of the spacer between the -35 and -10 regions in sigmas promoter selectivity in Escherichia coli

In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.     

Specific recognition of the -10 promoter element by the free RNA polymerase sigma subunit

Bacterial RNA polymerase holoenzyme relies on its sigma subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the sigma subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free sigma, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with sigma region 1 playing a role in the masking. To analyze the DNA-binding properties of the free sigma, we selected single-stranded DNA aptamers that are specific to primary sigma subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by sigma region 2 occurs without major structural rearrangements of sigma observed upon the holoenzyme formation and is not inhibited by sigma regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated sigma subunit and a short aptamer oligonucleotide.     

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.     

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).