Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase σ Subunit

Abstract

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 ...

The core-independent promoter-specific interaction of primary sigma factor

“…The preferential binding of σ A , by itself, to the promoter −10 (TATAAT) but not −35 element in vitro (Figures 2B, C and 6C) is consistent with the findings that the consensus sequences of GTA(C/T)AATGGGA and TGTAGAAT, both of which contain the TATAAT-like sequence, are required for single stranded deoxyribonucleic acid (ssDNA) aptamer binding to the Thermus aquaticus free σ A (54,55). It is also consistent with our observation that the −10 specific promoter interaction of σ A is unable to allow the σ A to discern the optimal promoter spacing (Figure 6).…”

Structural Basis for Promoter −10 Element Recognition by the Bacterial RNA Polymerase σ Subunit

“…Optimal binding of RNAP holoenzyme to fork-junction promoter probes, which have a mostly single-stranded –10 element, required the base-pair at –12 (Guo and Gralla, 1998), suggesting that the –12 position normally remains base-paired even in RP o (Figure 1A). Nevertheless, T -12 can also be recognized in the context of ssDNA (Feklistov et al, 2006; Roberts and Roberts, 1996; Sevostyanova et al, 2007). …”

“…Holoenzyme sequence-specifically binds single-stranded DNA (ssDNA) corresponding to the –10 element nontemplate-strand (nt-strand) sequence (Marr and Roberts, 1997; Roberts and Roberts, 1996; Savinkova et al, 1988), and this activity has been mapped to structural domain 2 of σ (σ 2 ; (Severinova et al, 1996; Young et al, 2001). Free σ, or fragments containing σ 2 , have sequence-specific ssDNA binding activity for the nt-strand –10 element sequence (Feklistov et al, 2006; Sevostyanova et al, 2007; Zenkin et al, 2007). In fact, the essence of RNAP promoter melting activity is localized to nt-strand – 10 element/σ 2 contacts (Young et al, 2004).…”

DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ D Proteins

“…Disulfide cross-linking suggests that a similarly compact conformation predominates in solution (25). Conversely, other studies support the idea that factors may specifically recognize elements of the promoter even in the absence of core RNA polymerase (12,16,23). We suggest that there is an equilibrium between the compact con- (Table 1), promoter-lacZ fusions were generated from the indicated upstream endpoints ({) and either of the two downstream endpoints, designated a and b (}).…”

“…Despite recent progress in RNAP structural biology (reviewed in reference 20), we still lack a high-resolution view of key transcription intermediates in open-complex formation. Development of simplified model systems is one promising approach for dissecting the interactions that occur during transcription initiation (10,23,29).…”