Regulatory sequences in sigma 54 localise near the start of DNA melting

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Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (␣ 2 ␤␤; E) associates with a sigma () subunit to form the holoenzyme (E Multisubunit DNA-dependent RNA polymerases (RNAP) 1 are complex molecular machines that synthesize a RNA copy from a DNA template. In Escherichia coli, five subunits (␣ 2 ␤␤Ј) form the RNAP catalytic core (E) that associates with a sigma () subunit to form the holoenzyme (E). The subunit imparts on the core RNAP the ability to specifically recognize and initiate transcription from promoters. Biochemical and structural studies indicate that the protein-protein contacts at the interface between core RNAP and , an extensive and functionally specialized sets of surfaces, govern the conformational changes that allow efficient promoter recognition and transcription initiation (1-3).Sequence comparisons reveal two unrelated families of RNAP factors. Members of the major family of factors form RNAP holoenzymes that recognize promoters and form transcriptionally competent promoter complexes in the absence of other factors or energy sources. This family, which includes most bacterial factors is named after the prototypical housekeeping of E. coli, 70 . Members of the second, minor family of factors, the 54 family, form RNAP holoenzymes that recognize promoters but require additional protein factors and a source of energy in the form of ATP or GTP hydrolysis for formation of transcriptionally competent promoter complexes (4 -6). Despite the differences in pathways that lead to transcription-competent open promoter complexes, both classes of factors occupy similar positions within their respective RNAP holoenzymes and appear to utilize some common RNAP surfaces for transcription initiation (7-10).Binding of a 70 family factor induces conformational changes within the core RNAP (2,11,12). Structural modules of the core RNAP, designated as the ␤Ј clamp, the ␤ flap, and the ␤ lobes, interact with a 70 family subunit ( A ) in the structures of Thermus aquaticus and Thermus thermophilus RNAP holoenzymes and undergo conformational changes, which orientate and position 70 DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3). The importance of these conformational changes is underlined by our recent observation that removal of the E. coli RNAP ␤ flap domain abolished the ability of the mutant E 70 to recognize promoters of the Ϫ10/Ϫ35 class (13). E 54 recognizes and binds promoters containing conserved consensus elements centered around Ϫ24 and Ϫ12 nucleotides upstream of the transcription start site at ϩ1. These promoter elements can be considered as functional analogues of the Ϫ35/Ϫ10 consensus promoter elements recognized by E 70 cla...

Section: Discussionmentioning

confidence: 99%

Section: Organization Of the Regulatory Center Within E 54 Closed Commentioning

confidence: 99%

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confidence: 99%

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Multiple Roles of the RNA Polymerase β Subunit Flap Domain in ς54-Dependent Transcription

Wigneshweraraj

Kuznedelov²,

Severinov³

et al. 2003

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“…1 for AAAϩ domain features) of the 54 activator PspF (PspF ) is necessary and sufficient to activate transcription of the 54 -RNAP in vitro and in vivo (14,15). The 54 activator signature sequence GAFTGA loop 1 directly binds to the region I of 54 , which is involved in maintaining a closed 54 -RNAP promoter complex (16,17 (19,20). Signaling between Walker motif B and the GAFTGA loop 1 involves tight interactions between the PspF Walker B residue Glu 108 and Asn 64 , nucleotide bindingdependent motions of linker regions 1 and 2, and a number of signaling residues that would position the GAFTGA loop 1 to contact 54 .…”

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Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Schumacher

Joly

Rappas

et al. 2007

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Transcriptional initiation invariably involves the transitionRegulation of transcription enables cells to adapt and differentiate through coordination of protein synthesis. Two major mechanisms exist to control gene transcription as follows: one through recruitment to or preventing the RNA polymerase (RNAP) 4 binding to promoter DNA and one through activation or inhibition of the RNA polymerase activity. Despite the variations in subunit numbers that constitute the basal transcription machinery, RNA polymerases are structurally and functionally highly conserved in all kingdoms of life (1). During the initiation of transcription, RNA polymerases invariably melt the promoter DNA and have to engage the single-stranded template DNA in the structurally conserved catalytic cleft (2).The major variant bacterial 54 -RNAP is regulated by an activating mechanism with some resemblance to the operation of eukaryotic RNAP II. In both cases, passing from the closed to the open RNAP promoter complex requires activator proteins that hydrolyze nucleoside triphosphate to drive open RNAP promoter complex formation (3). The 54 -RNAP binds to specific promoter sites centered on positions Ϫ24 and Ϫ12 relative to the transcription start site and remains in a transcriptionally silent conformation. Open complexes of the 54 -RNAP promoter complexes are thermodynamically labile, and activation relies on the productive coupling of nucleoside triphosphate hydrolysis (energy coupling) from 54 activators to the 54 -RNAP promoter complex. The 54 activator-dependent initial events in open complex formation are mediated by structural changes between 54 and the promoter DNA, which can be melted from position Ϫ12 to Ϫ5 in a reaction that can occur independently from RNAP core determinants. The restructuring of promoter DNA by activated 54 has been termed 54 isomerization (4 -6). Following the initial events and requiring the presence of the RNAP core, the promoter DNA opening extends to position ϩ3 relative to the transcription start site (7).Energy coupling primarily involves intimate interactions between the activator and the 54 promoter complex. 54 activators, also termed enhancer-binding proteins (EBP), belong to the versatile AAAϩ protein (ATPases associated with various cellular activities) family of molecular machines (for review see Refs. 8 -10). Members of EBPs include the well studied NtrC, PspF, DctD, XylR, NifA, and DmpR proteins (11). PspF is the phage shock protein F that activates transcription of psp genes involved in the phage shock response in Escherichia coli (12, * This work was supported in part by the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. Tel.: 44-207-5945366; Fax: 44-207-5842056; E-mail: j.schumacher@imperial.ac.uk. 2 Recipient of EMBO F...

“…However, 54 -RNAP footprints the Ϫ12 GC-region differently from 54 , suggesting that core RNAP changes 54 -promoter interactions in some way (26). The protein-DNA arrangement at the Ϫ12-position, termed the regulatory center (17,27), undergoes conformational changes during activation.…”

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Mapping σ54-RNA Polymerase Interactions at the –24 Consensus Promoter Element

Burrows

Severinov

Ishihama

et al. 2003

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The 54 promoter specificity factor is distinct from 70 -type factors. The 54 -RNA polymerase binds to promoters with conserved sequence elements at ؊24 and ؊12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between 54 -RNA polymerase and promoter DNA is poorly characterized, contrasting with 70 . Here, 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of 54 is close to and may therefore interact with the consensus ؊24 promoter element. We show that the spatial relationship between the 54 -RNA polymerase and the ؊24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between 54 -RNA polymerase and the consensus ؊12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some ؊12 promoter region-54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the ؊12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the ␤-subunit flap domain contributes to different sets of -promoter DNA interactions at 54 -and 70 -dependent promoters.Transcription of DNA is a fundamental process needed for regulation of cellular adaptation and differentiation and is carried out by DNA-dependent RNA polymerases (RNAPs).