The bacterial enhancer-dependent RNA polymerase.

Biochemical Journal (2016) 473 3741–3753 DOI: 10.1042/BCJ20160741C Review Article The bacterial enhancer-dependent RNA polymerase Nan Zhang1,2, Vidya C. Darbari3,4, Robert Glyde3, Xiaodong Zhang3 and Martin Buck1 1 Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K.; 2Neuroscience Research Program, Houston Methodist Research Institute, 6670 Bertner Avenue, Houston, TX 77030, U.S.A.; 3Division of Macromolecular Structure and Function, Imperial College London, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K.; and 4School of Biological and Chemical Sciences, Department of Chemistry and Biochemistry, Queen Mary University of London, Mile End Road, London E1 4NS, U.K. Correspondence: Martin Buck () or Xiaodong Zhang () Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript. Introduction Received: 8 August 2016 Revised: 22 August 2016 Accepted: 25 August 2016 Version of Record published: 27 October 2016 Gene transcription is a pervasive and fundamental process necessary for accessing information in genomes. Transcription of DNA by RNA polymerases is a highly regulated process, underpinning cellular decisions in adaptation and differentiation. RNA polymerases range in complexity from the simpler single subunit phage encoded enzymes to the multi-subunit enzymes found in all three kingdoms of life. All initiate transcription from promoters to achieve patterns of gene-specific expression. Promoter specificity factors directing RNA polymerases to promoters in bacteria fall into two classes, with the sigma54 class requiring specialized AAA+ transcription activator proteins, the bacterial enhancer-binding proteins (bEBPs), for its regulation, whereas the sigma70 class can function in transcription initiation without additional control proteins [1–5]. A range of genetic and biochemical studies have established some special properties of the sigma54 containing RNA polymerase, which distinguish it from the sigma70-type holoenzyme, and which can be attributed directly to the sigma factor itself. These include the ability of the sigma54 factor in the absence of the core RNA polymerase to bind promoter DNA and to be remodelled by its cognate bEBPs in an ATPase reaction, and equivalent activities are not evident with the sigma70 factor [6–20]. The lack of any significant sequence similarity between sigma54 and sigma70 was noted early on and is borne out by their very different molecular structures and differing modes of action within the core RNA polymerase as described below. © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND). 3741 Biochemical Journal (2016) 473 3741–3753 DOI: 10.1042/BCJ20160741C Structural studies Until recently, the only high-resolution structural information for bacterial RNAP holoenzymes containing a sigma factor was that for those bound by the primary sigma70-type factors (as recently reviewed in [21]). These structures provide detailed mechanistic insights into promoter recognition and transcription initiation by the primary sigma factors and also the group 2 and ECF sigma factors, which are all related within the sigma70 class. A detailed structure-based mechanistic understanding has been missing for sigma54-dependent transcription initiation and could not be deduced due to the lack of sequence and inferred structural homology of sigma54 with the primary sigma factors [4,22]. Cryo-EM reconstructions of σ54-RNAP holoenzyme in the apo-form and bound to an Escherichia coli AAA+ transcription activator protein domain (from PspF, a wellstudied bEBP), provided at medium resolution an initial snapshot of the sigma54 holoenzyme in the closed and one nucleotide-dependent activator engaged state. Additionally, NMR structures of the sigma54 corebinding domain and the domain containing the −24 promoter element-binding RpoN box from Aquifex aeolicus were obtained in the group of Wemmer and colleagues [6,17,23–25]. However, defining in structural terms the precise interfaces that would exist between sigma54, the RNA polymerase core enzyme and the bEBPs is necessary to help establish its mode of action and ATPase dependence. In particular, a range of functional states of the transcription complex along the transcription initiation pathway need to be structurally defined. Sigma70 contains four major functional regions: region 1 (σ1.1) which is located in the downstream DNA channel in the apo-form where promoter DNA is not fully engaged, but is ejected from the channel in the RNAP–promoter DNA complex [26,27]; regions 2 and 3 (σ2 and σ3) which are the major core enzyme-binding domains (CBDs) with σ2 also playing an important role in promoter melting through intercalation with DNA bases [28]; and region 4 (σ4) which is responsible for −35 promoter recognition [29]. The sigma54 can be divided into three regions based on sequence and function, although they have varying degrees of functional similarities to the regions of sigma70 and no sequence similarity (Figure 1A,B). In sigma54, Region I (RI, residues 1–56, E. coli numbering) helps maintain the closed promoter complex by inhibiting the DNA melting reaction, includes the major binding site for the bEBPs, and directs the formation of a DNA fork junction structure at the base pair immediately downstream of the promoter −12 element [19,2 (...truncated)