Bacterial enhancer-binding proteins: unlocking σ54-dependent gene transcription

Centre for Structural Biology, Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK.
Current Opinion in Structural Biology (Impact Factor: 7.2). 03/2007; 17(1):110-6. DOI: 10.1016/
Source: PubMed


Bacterial transcription relies on the binding of dissociable sigma (sigma) factors to RNA polymerase (RNAP) for promoter specificity. The major variant sigma factor (sigma54) forms a stable closed complex with RNAP bound to DNA that rarely spontaneously isomerises to an open complex. ATP hydrolysis by bacterial enhancer-binding proteins is used to remodel the RNAP-sigma54-DNA closed complex. Recently, a wealth of structural information on bacterial enhancer-binding proteins has enabled unprecedented insights into their mechanism. These data provide a structural basis for nucleotide binding and hydrolysis, oligomerisation and the conversion of ATPase activity into remodelling events within the RNAP-sigma54 closed complex, and represent advances towards a complete understanding of the sigma54-dependent transcription activation mechanism.

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    • "In contrast to E␴ 70 , the alternate ␴ 54 -RNAP holoenzyme (E␴ 54 ), which recognizes promoters marked by the −24 (GG) and −12 (TGC) consensus sequences upstream of the transcription start site, binds promoter sequences in an energetically favorable closed conformation that rarely isomerizes into an open complex (Buck et al., 2000; Wigneshweraraj et al., 2008). To initiate transcription , E␴ 54 needs to bind specialized activator proteins (known as bacterial enhancer-binding proteins [bEBP]) that hydrolyze ATP and use the energy released to remodel their substrates (Rappas et al., 2007; Schumacher et al., 2004; Xu and Hoover, 2001; Zhang et al., 2002) (Fig. 1D). Thus, ␴54 is structurally different from ␴70 members, and is composed of three regions based on function: the N-terminal region I interacts with activator proteins and the −12 promoter element, where DNA melting originates; the central region II is variable and sometimes absent; the C-terminal region III contains a number of functional modules, including determinants that bind core RNAP and promoter DNA (Bordes et al., 2004; Bose et al., 2008a,b; Buck et al., 2000; Chaney et al., 2001; Gallegos et al., 1999). "
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    ABSTRACT: Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure-function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: i.) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; ii.) the occurrence in nature of "mutant-type" or duplicated rifampicin-resistant RNA polymerases; iii.) the RNA polymerase genetic engineering method for strain improvement and drug discovery. Copyright © 2014. Published by Elsevier B.V.
    Journal of Biotechnology 12/2014; 202. DOI:10.1016/j.jbiotec.2014.11.024 · 2.87 Impact Factor
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    • "FlrA is a σ 54-dependent enhancer-binding protein (EBP) that contains an Nterminal receiver domain, central ATPase Associated with diverse cellular Activities (AAA+) domain, and a C-terminal DNA-binding domain. σ54-dependent EBPs typically bind 100–1000 bp upstream of the −12/−24 σ54 promoter (Rappas et al., 2007). Upon activation by phosphorylation or dephosphorylation of the REC domain, these proteins oligomerize into ring-shaped structures which loop the DNA and activate transcription. "
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    ABSTRACT: Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homolog of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.
    Molecular Microbiology 10/2013; 90(6). DOI:10.1111/mmi.12432 · 4.42 Impact Factor
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    • "Analysis of the 458 bp promoter sequence using the search algorithms GenomeMatScan and TRES, failed to identify palindromic or inverted repeat regions, typical of XylR/NtrC family enhancer binding proteins, (EBPs) [19,26]. EBPs are reportedly essential for transcriptional activation of σ54 promoters and facilitate the integration of promoter activation with host signal responses to environmental cues and physiological states, [27,28]. Comparative analysis of the paaL promoter with 9 other predicted σ54 promoter sequences from P. putida KT2440, was carried out using the Multiple Em for Motif Elucidation algorithm, MEME [29]. "
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    ABSTRACT: Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ54 consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.
    BMC Microbiology 10/2011; 11(1):229. DOI:10.1186/1471-2180-11-229 · 2.73 Impact Factor
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