Structural Modules of RNA Polymerase Required for Transcription from Promoters Containing Downstream Basal Promoter Element GGGA

Abstract

We recently described a novel basal bacterial promoter element that is located downstream of the ؊10 consensus promoter element and is recognized by region 1.2 of the subunit of RNA polymerase (RNAP). In the case of Thermus aquaticus RNAP, this element has a consensus sequence GGGA and allows transcription initiation in the absence of the ؊35 element. In contrast, the Escherichia coli RNAP is unable to initiate transcription from GGGA-containing promoters that lack the ؊35 element. In the present study, we demonstrate that subunits from both E. coli and T. aquaticus specifically recognize the GGGA element and that the observed species specificity of recognition of GGGA-containing promoters is determined by the RNAP core enzyme. We further demonstrate that transcription initiation by T. aquaticus RNAP on GGGA-containing promoters in the absence of the ؊35 element requires region 4 and C-terminal domains of the ␣ subunits, which interact with upstream promoter DNA. When in the context of promoters containing the ؊35 element, the GGGA element is recognized by holoenzyme RNAPs from both E. coli and T. aquaticus and increases stability of promoter complexes formed on these promoters. Thus, GGGA is a bona fide basal promoter element that can function in various bacteria and, depending on the properties of the RNAP core enzyme and the presence of additional promoter elements, determine species-specific differences in promoter recognition.In bacteria, recognition of promoters is accomplished through specific interactions of the RNA polymerase (RNAP) 3 holoenzyme with consensus elements of promoter DNA. Most housekeeping promoters are recognized by RNAP holoenzyme containing the major subunit (called 70 in Escherichia coli or A in other bacteria). The Ϫ10 (TATAAT) and the Ϫ35 (TTGACA) consensus elements of these promoters interact with conserved regions 2.4 and 4.2 of , respectively (1). A subset of promoters (the so-called extended Ϫ10 promoters) contain a TG motif one nucleotide upstream of the Ϫ10 element that is recognized by region 2.5 of ; these promoters do not require the Ϫ35 element for their activity (2). Some strong promoters contain an A/T-rich UP element that is located upstream of the Ϫ35 element and is specifically recognized by the C-terminal domains of the RNAP ␣ subunits (␣CTDs) (3).In addition to specific interactions with conserved promoter elements, nonspecific RNAP-DNA interactions also contribute to promoter recognition. In particular, it was shown that nonspecific contacts of E. coli RNAP ␣CTDs with upstream DNA play an important role in promoter recognition even in the absence of the UP element (4 -6) and that contacts of 70 region 4 with DNA stimulate recognition of extended Ϫ10 promoters lacking the Ϫ35 element (7).Sigma subunits of the 70 class are unable to recognize promoters in the absence of the RNAP core. We have recently demonstrated that isolated A from thermophilic bacterium Thermus aquaticus can recognize single-stranded DNA aptamers (sTaps, for sigma T. aquaticus aptamers) that cont...

RNA polymerase-promoter interactions determining different stability of the Escherichia coli and Thermus aquaticus transcription initiation complexes

“…Consistent with a previous report (10), our data indicate that Taq RNAP is not compromised in terms of its intrinsic affinity to the single-stranded non-template segment of transcription bubble bearing the −10 element. However, in agreement with the known biochemical results (32), we found that the dependence of RNAP affinity on the sequence of discriminator is different in Taq and E. coli RNAPs: the ratio of affinities of oligos 3 and 4 bearing, respectively, the GGGA and CCCT discriminator sequences is 150 for Taq Eσ A and only 7 for to Ec Eσ 70 .…”

“…The substitutions caused considerable, ∼100-fold, change in affinity to Taq Eσ A , whereas only ∼10-fold change was observed with Ec Eσ 70 (Figure 4A, Table 1). These data correlate well with reported effects caused by similar sequence changes on efficiencies of transcription initiation by these enzymes, suggesting that the downstream promoter motif is more important for Taq RNAP than for E. coli RNAP (32). …”

A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing

“…Taq RNAP mutants with deletions of the β flap tip and αCTDs are unable to efficiently recognize −10/−35 promoters (24). Thus, to test termination by these RNAP mutants we used a DNA template that contained an extended −10 promoter, gal P1, fused to the tR2 terminator.…”

Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation

“…Thus, nonconserved substitutions in region 1.2 in Taq A may affect the open complex stability through weakening the -DNA contacts. Importantly, however, in contrast to the previously studied alanine substitutions in 70 , A -specific substitutions do not disrupt interactions of region 1.2 with DNA, because A region 1.2 was shown to specifically recognize the GGGA element downstream of the Ϫ10 element in promoter complexes of Taq RNAP (10,18).…”

“…1A), incompatible with double-stranded DNA conformation, explaining previously established roles of region 2 in promoter recognition, DNA melting, and stabilization of RNAP-DNA interactions (1,(13)(14)(15)(16)(17). Although the exact position of the nontemplate DNA strand downstream of the Ϫ10 element remains unknown, structural modeling, site-specific cross-linking, and biochemical analyses suggest that this DNA segment may directly contact region 1.2, depending on the DNA sequence context (10,11,18,19). Amino acid substitutions at various positions of region 1.2 in Eco 70 were shown to decrease RNAP activity, inhibit promoter DNA melting, and destabilize the open promoter complex (Fig.…”