Bacterial enhancer-binding proteins: unlocking sigma54-dependent gene transcription.
ABSTRACT 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.
SourceAvailable from: Daniela Pasanisi[Show abstract] [Hide abstract]
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; DOI:10.1016/j.jbiotec.2014.11.024 · 2.88 Impact Factor
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ABSTRACT: Lysine 2,3-aminomutase (KAM, EC 126.96.36.199.) catalyzes the interconversion of L-lysine and L-β-lysine. The transcription and regulation of the kam locus, including lysine-2,3-aminomutase-encoding genes in B. thuringiensis were analyzed in this study. RT-PCR analysis revealed that this locus forms two operons: yodT (yodT-yodS-yodR-yodQ-yodP-kamR) and kamA (kamA-yokU-yozE). The transcriptional start sites (TSSs) of the kamA gene were determined using 5'-RACE analysis. A typical -12/-24 Sigma(54) binding site was identified in the promoter PkamA, which is located upstream of the kamA gene TSS. A β-galactosidase assay showed that PkamA, which directs the transcription of the kamA operon, is controlled by the Sigma(54) factor and is activated through the Sigma(54)-dependent transcriptional regulator KamR. The kamA operon is also controlled by Sigma(K) and regulated by the GerE protein in the late stage of sporulation. The kamR and kamA mutants were prepared by homologous recombination to examine the role of the kam locus. The results showed that the sporulation rate in HD(ΔkamR) was slightly decreased compared to that in HD73, whereas that in HD(ΔkamA) was similar to that in HD73. It means that other genes regulated by KamR are important for sporulation.Journal of Bacteriology 06/2014; DOI:10.1128/JB.01675-14 · 2.69 Impact Factor
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ABSTRACT: σ54-dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to σ70, the σ54- RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerizes into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activity of activator proteins that bind remotely upstream of the transcriptional start site. These activators belong to the large AAA protein family and the majority of them consist of an N-terminal regulatory domain, a central AAA domain and a C-terminal DNA binding domain. Here we use a functional variant of the NorR activator, a dedicated NO sensor, to provide the first structural and functional characterization of a full length AAA activator in complex with its enhancer DNA. Our data suggest an inter-dependent and synergistic relationship of all three functional domains and provide an explanation for the dependence of NorR on enhancer DNA. Our results show that NorR readily assembles into higher order oligomers upon enhancer binding, independent of activating signals. Upon inducing signals, the N-terminal regulatory domain relocates to the periphery of the AAA ring. Together our data provide an assembly and activation mechanism for NorR.Molecular Microbiology 10/2014; DOI:10.1111/mmi.12844 · 5.03 Impact Factor