During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

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...

Bacterial RNA polymerase holoenzyme relies on its subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the subunit are positioned at an optimal distance to allow specific recognition of the ؊10 and ؊35 promoter elements, respectively. In free , the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with region 1 playing a role in the masking. To analyze the DNA-binding properties of the free , we selected single-stranded DNA aptamers that are specific to primary 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 region 2 occurs without major structural rearrangements of observed upon the holoenzyme formation and is not inhibited by 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 subunit and a short aptamer oligonucleotide. Recognition of the Ϫ10 element can be modeled in a complex of holo-RNAP with short oligonucleotides corresponding to the nontemplate promoter strand and containing the Ϫ10 sequence (nontemplate oligonucleotides) (6 -9). However, despite playing a crucial role in promoter recognition by holo-RNAP, free is unable to specifically recognize either promoters or nontemplate oligonucleotides (1, 10 -12).Crystal structures of holo-RNAPs from Thermus aquaticus and Thermus thermophilus revealed that contains three domains (2, 3, and 4, named after corresponding conserved regions) connected by flexible linkers (13,14). In holoenzyme, these domains are spread on the core enzyme surface, with 2 and 4 being positioned at an optimal distance relative to each other to allow interactions with the Ϫ10 and Ϫ35 promoter elements. The structure of the isolated individual domains of is almost identical to their structures in holo-RNAP (13-16). However, the structure of the full-length subunit in a free state remains unknown.Based on indirect biochemical and biophysical data, several mechanisms of inhibition of DNA-binding activity in free have been proposed. First, the N-terminal region of (region 1.1) was shown to inhibit DNA binding. Deletion of this region allows 70 to weakly bind double-stranded promoter DNA with some specificity (17, 18) but does not allow the recognition of single-stranded nontemplate oligonucleotides by free (15,19). It was proposed that region 1.1 may inhibit DNA binding by (i) direct masking of region 4 (17, 18), (ii) direct interactions with region 2 (20, 21), or (iii) by an indirect allosteric mechanism (22).Second, it was shown that free adopts a compact conformation in which DNA-recognition domains 2 and 4 are brought closer to each other than in the holoenzyme (23). The sub-optimal positioning of 2 and 4 is likely to interfere with simultaneous recognition of the Ϫ10 and Ϫ35 elements. Furthermore, the ...

RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase beta subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20 degrees C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2-3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.

Highly conserved amino acid residues in region 2 of the RNA polymerase subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.Promoter opening is a temperature-dependent process that requires conformational changes in both RNA polymerase (RNAP) and DNA (4). In bacteria, promoter recognition and opening are accomplished by the RNAP holoenzyme, which consists of a core enzyme and a specificity factor, the subunit. DNA melting is initiated through specific interactions of conserved region 2 of the subunit with the Ϫ10 promoter element (consensus sequence, TATAAT) (7). Genetic and biochemical analyses have implicated conserved amino acids from different subregions of region 2 both in interactions with core RNAP and in DNA melting. Mutations of conserved residues in subregions 2.1 and 2.2 affected -core interactions and defined this part of region 2 as the main core binding site of ( Fig. 1A) (20). Mutations in a cluster of highly conserved aromatic residues in subregion 2.3 of Escherichia coli 70 and Bacillus subtilisA were shown to lead to severe defects in promoter opening that could be partially suppressed by an increase in the temperature (Fig. 1A) (6, 9, 16). It was therefore proposed that these amino acids may directly initiate DNA melting through hydrophobic interactions with nucleotide bases of the Ϫ10 element. Particular attention has been paid to Y430 and W433 (E. coli numbering), which were proposed to interact with a conserved adenine at the second position of the Ϫ10 element (position Ϫ11A) in the nontemplate DNA strand (reference 18 and references therein). Mutations of other conserved residues in subregions 2.2, 2.3, and 2.4 (including basic amino acids in subregion 2.3) also had dramatic effects on promoter opening (Fig. 1A). These amino acids were proposed to play a role in the recognition and/or proper positioning of promoter DNA (6, 7, 21). At the same time, the detailed mechanism of promoter melting remains unknown due to the absence of information on the high-resolution structure of the open promoter complex.In RNAPs from different species there are certain functional differences in promoter recognition and opening which are believed to result, at least in part, from their different adaptations (1, 3). In particular, RNAPs from thermophilic bacteria have long been known to melt DNA and initiate transcription at higher temperatures than their mesophilic counterparts (12,13,15,17,24). In accordance with this, we recently demonstrated that RNAP from the thermophilic bacterium Thermus aquaticus opens promoters at temperatures above 37°C but, unlike RNAP from E. coli, is unable to open promoters at lower temperatures (10). Surprisingly, we found that RNAP from the mesophilic organism Deinococcus radiodurans, which is closely related phylogenetically to T. aquaticus, had similar cold sensitivity of promoter opening, implying that this prope...