The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70

Abstract

The "" subunit of prokaryotic RNA polymerase allows genespecific transcription initiation. Two families have been identified, 70 and 54 , which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the 70 -type factors have been well characterized structurally by x-ray crystallography, no high resolution structural information is available for the 54 -type factors. Here we present the NMR-derived structure of the C-terminal domain of 54 from Aquifex aeolicus. This domain (Thr-323 to Gly-389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the 70 -type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. The homology of this structure with other DNA-binding proteins, combined with previous biochemical data, suggests how the C-terminal domain of 54 binds to DNA.Transcription, the synthesis of RNA from double-stranded DNA, is a fundamental process in all forms of life. The primary protein complex catalyzing transcription is composed of five subunits, ␣ 2 ␤Ј␤, called "core RNA polymerase" (RNAP).2 Core RNAP is fully competent to synthesize RNA from DNA. However, the initiation of transcription at specific DNA sequences requires additional protein(s). In bacteria, these additional proteins are called the " factors" (1, 2). The factor facilitates transcription at specific DNA sequences by 1) binding to the core RNAP to form the -RNAP holoenzyme, 2) recognizing and binding to a specific DNA sequence next to the transcription start site, called the promoter element, and 3) opening the double-stranded DNA to initiate transcription.Based on protein sequence homology, factors can be grouped into two classes, 70 and 54 , which were named by the molecular weights of the first members identified (reviewed in Ref. 1 is involved in regulating the kinetics of transcription initiation; 2 binds to the Ϫ10 promoter element and is essential for melting the DNA;3 stabilizes the open complex formation by binding the extended Ϫ10 element; and 4 binds specifically to the Ϫ35 promoter element (reviewed in Ref. 17).In contrast to 70 , 54 is divided into three regions based on function (18 -20). Region 1 (E. coli-(1-55)) interacts with the upstream activator protein to control promoter melting; region 2 (E. coli-(70 -304)) contains the minimal RNAP-binding domain (E. coli-(70 -180) and a part that enhances DNA binding affinity (E. coli-(180 -304)); region 3 (E. coli-(329 -477)) recognizes and binds the consensus promoter elements. This C-terminal part of the protein contains a region that crosslinks to DNA (E. coli-(329 -346)), a predicted helix-turn-helix motif (E. coli-(366 -386); Fig. 1), and a highly conserved sequence (E. coli-* This work was supported by National Institutes of Health Grant GM62163 (to D. E. W.) and by a University of Calif...

Crystal structure of Aquifex aeolicus σ N bound to promoter DNA and the structure of σ N -holoenzyme

“…Structural information on σ N was limited to NMR structures of isolated domains [CBD (18) and RpoN domain (14,17)] and a 3.8-Å resolution crystal structure of Eσ N that revealed the overall disposition of the σ N domains in complex with RNAP but is shown here to contain significant errors in detail (Figs. S3-S5 and SI Appendix, Tables S2 and S3).…”

“…High-resolution NMR structures of the core-binding domain (CBD; Fig. 1B), the RpoN domain, and the RpoN domain in complex with −24 element DNA were determined using Aae σ N domains (14,17,18). The DNA templates for most of our biochemical and structural studies were based on the Aae dhsU promoter (16), which corresponds to the consensus σ N promoter (Fig.…”

Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

“…The C-terminal domain allows specific binding of the activator to a particular UAS (Doucleff et al, 2005b), limiting the activity of the EBP to a few promoters in the chromosome; this fact allows the s 54 factor to be involved in the transcription of different non-related pathways in the same bacterium. Functional EBPs without an HTH motif have been described in Helicobacter pylori, Chlamydia trachomatis and Rhodobacter sphaeroides (Brahmachary et al, 2004;Poggio et al, 2005;Beck et al, 2007).…”

“…Within these regions a distinctive signature of the EBPs is the GAFTGA sequence that is located in the C3 region; experimental evidence suggests that this sequence interacts with Es 54 during ATP hydrolysis (Chaney et al, 2001; De Carlo et al, 2006). For some activators such as DctD, NifA and PspF, it has been shown that, in the absence of the N-and C-terminal domains, the central region by itself is sufficient to activate transcription (Xu et al, 2004).The C-terminal domain allows specific binding of the activator to a particular UAS (Doucleff et al, 2005b), limiting the activity of the EBP to a few promoters in the chromosome; this fact allows the s 54 factor to be involved in the transcription of different non-related pathways in the same bacterium. Functional EBPs without an HTH motif have been described in Helicobacter pylori, Chlamydia trachomatis and Rhodobacter sphaeroides (Brahmachary et al, 2004;Poggio et al, 2005;Beck et al, 2007).…”

Evidence for self‐association of the alternative sigma factor σ54

“…Region III (amino acids 121-477) has functional modules that include determinants that are able to bind core RNAP (amino acids 120-215) and promoter DNA (amino acids 329-463). In particular, the C-terminal module contains a sub-region (amino acids 329-346) that may be cross-linked to DNA and a signature motif for r 54 proteins called an RpoN box (amino acids 454-463) [12,13], which has been shown to contain a classical helix-turn-helix module that is able to bind to the À24 promoter elements [14,15]. In r…”