Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In herpes simplex virus type 1 (HSV-1)-infected HEp-2 cells, amanitin added before or at various times after infection always reduced viral multiplication. Also, the three waves of transcription of HSV-1 DNA, which led to the synthesis of alpha, beta-, and gamma-polypeptides, were all sensitive to amanitin in HEp-2 cells, and the amanitin-sensitive RNA polymerase activities of isolated nuclei were equally sensitive to the inhibitor before and during the infection. On the contrary, HSV-1 DNA transcription was totally unaffected by amanitin in AR1/9-5B cells, a mutant subline of CHO cells that possesses an amanitin-resistant RNA polymerase B. Together, these results strongly suggest that HSV-1 DNA utilizes for its transcription a polymerase undistinguishable from host cell RNA polymerase B with respect to its sensitivity to amanitin.
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PMID:Evidence that herpes simplex virus DNA is transcribed by cellular RNA polymerase B. 19 58

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase. The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters. AR2 but not AR1 is essential for activation at all the Class II promoters that were tested. The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the -35 hexamer sequence. Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP- dependent promoter pmelRcon. At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex.
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PMID:Transcription activation at class II CRP-dependent promoters: the role of different activating regions. 901 61

The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.
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PMID:Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12. 972 23

The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the -45 region (CAP binding is centered at -61.5; the -45 region extends from -50 to -38). Certain mutations within the -45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These -45 region "UP mutants" are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a -45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the alpha-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the alpha-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold invariable CAP stimulation appears to depend on the -45 region sequence as our -45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.
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PMID:CAP, the -45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli. 973 18

The promoter of the FNR-activated yfiD gene of Escherichia coli has an unusual architecture because it contains two FNR sites, an arrangement usually associated with FNR-mediated repression. Investigation of yfiD promoter derivatives with altered FNR sites revealed that occupation of the far upstream FNR site (FNR II) downregulated expression, despite the presence of a FNR dimer activating expression from the promoter proximal site (FNR I). Transcript mapping by primer extension, and mutagenesis of potential -10 elements, indicated that yfiD expression is driven from a single FNR-dependent promoter with FNR sites at -40.5 (FNR I) and -93.5 (FNR II). However, yfiD mRNA is processed in stationary-phase cultures independently of rne, rpoS, ihfA and fis to yield transcripts lacking 12 and 21 bases from their respective 5' ends. Single amino acid substitutions (G74-->C, F92-->S, A95-->P, R184-->P, P188-->A or L193-->P) in the surface of FNR that contains activating region 1 (AR1 contacts the alpha-subunit of RNA polymerase to promote transcription activation) reduced the inhibitory effect of FNR at FNR II, indicating that this region of the protein may have a role in repression as well as activation. The FNR variant F92-->S was notable because, although it activated transcription of yfiD (two FNR sites), it was unable to activate transcription from model Class I and II promoters, which contain only a single FNR site.
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PMID:Downregulation of Escherichia coli yfiD expression by FNR occupying a site at -93.5 involves the AR1-containing face of FNR. 976 78

The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD. Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.
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PMID:Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon. 1085 86

In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR. The ability of FNR to activate transcription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR-AR1 and FNR-AR3. To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA. All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-AR1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from PnarG and PdmsA in vivo. Transcription assays of PdmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro. Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA-binding properties. The function of FNR-S187P and FNR-G85A was also measured in strains containing sigma70 mutants (sigma70-K593A, sigma70-R596A and sigma70-K597A) known to be impaired in FNR-dependent transcription activation. Of all of the combinations analysed, only FNR-G85 and sigma70-K597 showed a genetic interaction, supporting the notion that FNR-AR3 and sigma70 interact functionally in the process of transcription activation. Lastly, the transcription activation defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate. As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.
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PMID:FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3. 1111 16

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia. The anaerobic incorporation of oxygen-sensitive [4Fe 4S] clusters promotes dimerization, which in turn enhances DNA binding. Four potential iron ligands (C20, C23, C29 and C122) are essential for normal FNR activity in vivo. Three FNR variants (C20S, C23G and C29G) retained the ability to incorporate oxygen-sensitive [4Fe 4S] clusters and to bind target DNA with essentially unimpaired affinity, suggesting that their failure to function normally in vivo resides at a later stage in the signal transduction pathway. The C122 variant failed to assemble iron-sulphur clusters and to bind DNA. Second-site substitutions that partially restore activity to FNR(C20S) were generated by error-prone polymerase chain reaction and were located in the dimer interface, in the activating regions (AR1, 2 or 3) or close to C122. Substitutions at E47, R48, E123, I124, E127 or T128 allowed the extent of the FNR AR2 surface to be defined. Only one revertant, FNR(C20S Y69F G149S), specifically corrected the C20S defect. It was concluded that [4Fe 4S] cluster acquisition, dimerization and DNA binding are not sufficient to confer transcription regulatory activity on FNR: the iron-sulphur cluster must also be correctly liganded in order to establish effective activating contacts between FNR and RNA polymerase.
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PMID:Anaerobic acquisition of [4FE 4S] clusters by the inactive FNR(C20S) variant and restoration of activity by second-site amino acid substitutions. 1125 37

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.
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PMID:Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation. 1170 61

In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping.
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PMID:ToxR interferes with CRP-dependent transcriptional activation of ompT in Vibrio cholerae. 1195 6


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