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)

Influenza viruses were disrupted layer by layer with the nonionic detergent NP-40 at fixed pH. Treatment of the virions with NP-40 at neutral or mildly alkaline pH (6.8-8.0) yielded viral core structures containing M1 protein. The matrix M1 protein was selectively extracted from cores at acidic pH 3.0-4.5 with citrate, acetate, and phosphate buffers or with morpholinoethanesulfonic acid. The resulting M1 protein sedimented in a glycerol gradient with a coefficient of 2.8 S and most likely existed as a monomeric form of the 27,000-Da polypeptide. An antigenic map of the monomeric protein M1 tested with a panel of monoclonal anti-M1 antibodies was found to be similar to those of the assembled M1 protein in whole virions. The isolated M1 protein retained biological properties and inhibited the RNA polymerase activity of viral RNP. This transcription-inhibition function of M1 monomers was specifically restricted by one of the monoclonal antibodies studied.
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PMID:Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. 172 9

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
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PMID:Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa. 175 20

The base sequence of a specific DNA region identified as the promoter is investigated by means of the quantity Sr corresponding to "superdelocalizability" of oxygen ion of each phosphate for the ten DNA dimer units (XY/Y'X') and the six [(XY/Y'X') + H+] complexes. A mechanisum that the RNA polymerase can recognize its transcription site (phosphate), is proposed and applied to Escherichia coli promoters, lacUV5, recAp, rrnEp1, rrnEp2 (experimental facts).
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PMID:On the base sequences of the promoters in transcription initiation. 184 52

This paper describes the binding interactions of Escherichia coli transcription factors sigma 70 and NusA with core RNA polymerase, both free in solution and as a part of the functional transcription complex. High pressure liquid chromatography gel filtration and fluorescence techniques have been used to monitor the binding of these factors to core polymerase in solution at salt concentrations roughly comparable to the in vivo environment (250 mM-KCl, 50 mM-potassium phosphate (pH 7.5]; under these conditions all the interacting species exist separately as protein monomers. We find that sigma 70 and NusA binds competitively to core polymerase with a 1:1 binding stoichiometry in this milieu, and that NusA does not bind to the polymerase holoenzyme. Association constants of approximately 2 x 10(9) and 1 x 10(7) M-1 have been measured for the sigma 70-core polymerase interaction and for the NusA-core polymerase interaction, respectively. These findings are consistent with the original formulation of the NusA-sigma 70 cycle put forward by Greenblatt & Li, and provide the basis for a further (and preliminary) quantitative examination of these same interactions within the transcription complex. We use a number of molecular biological techniques, together with data from the literature, to estimate these binding constants in various phases of the transcription cycle. In keeping with our results in solution, we find that the effective binding affinity of sigma 70 for core polymerase within the "open" promoter-polymerase complex is at least 500-fold greater than that of NusA. As the transcription complex moves from the initiation to the elongation phase these relative binding affinities are reversed; the average association constant of NusA for the core polymerase in the elongation complex remains practically the same as in free solution (approx. 3 x 10(7) M-1), while the affinity of sigma 70 for core polymerase in this complex drops to less than 5 x 10(5) M-1. These results are used to begin to define the basic conformational states and interaction potentials of core polymerase in the various stages of the transcription cycle.
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PMID:Escherichia coli sigma 70 and NusA proteins. I. Binding interactions with core RNA polymerase in solution and within the transcription complex. 185 61

In Pseudomonas aeruginosa, the operon encoding tryptophan synthase (trpBA) is positively regulated by the TrpI protein and an intermediate in tryptophan biosynthesis, indoleglycerol phosphate (InGP). A gene fusion in which the trpBA promoter directs expression of the Pseudomonas putida xylE gene was constructed. By using a P. putida F1 todE mutant carrying this fusion on a plasmid, three cis-acting mutations that increased xylE expression enough to allow the todE strain to grow on toluene were isolated. The level of xylE transcript from the trpBA promoter was increased in all three mutants. All three mutations are base substitutions located in the -10 region of the trpBA promoter; two of these mutations make the promoter sequence more like the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. The activities of the wild-type and mutant trpBA promoters, as monitored by xylE expression, were assayed in P. putida PpG1 and in E. coli. The up-regulatory phenotypes of the mutants were maintained in the heterologous backgrounds, as was trpI and InGP dependence. These results indicate that the P. aeruginosa trpBA promoter has the key characteristics of a typical E. coli positively regulated promoter. The results also show that the P. aeruginosa and P. putida trpI activator gene products are functionally interchangeable.
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PMID:Up-promoter mutations in the trpBA operon of Pseudomonas aeruginosa. 190 57

We have developed an in vitro transcription system in which purified TrpI protein and indoleglycerol phosphate (InGP) activate transcription initiation at the trpBA promoter (trpPB) and repress initiation at the trpI promoter (trpPI) of Pseudomonas aeruginosa. The phenotypes resulting from mutations in the -10 region of both promoters indicate that the -10 region consensus sequence in P. aeruginosa is probably the same as that in Escherichia coli. Furthermore, in the absence of TrpI and InGP, the activities of the two promoters are inversely correlated: down mutations in trpPI lead to increased activity of trpPB, and up mutations in trpPB cause a decrease in trpPI activity. These results are a consequence of the fact that the two promoters overlap, so that RNA polymerase cannot form open complexes with both promoters simultaneously. Thus, in theory, by preventing RNA polymerase from binding at trpPI, TrpI protein could indirectly activate trpPB. However, oligonucleotide-induced mutations that completely inactivate trpPI do not relieve the requirement for TrpI and InGP to activate trpPB. Therefore, activation of trpPB is mediated by a direct effect of TrpI on transcription initiation at trpPB. In addition, the oligonucleotide-induced mutations in trpPI alter site II, the weaker of two TrpI binding sites identified in DNase I and hydroxyl radical footprinting studies (M. Chang and I. P. Crawford, Nucleic Acids Res. 18:979-988, 1990). Since these mutations prevent full activation of trpPB, we conclude that specific base pairs in site II are required for activation.
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PMID:Activation of the trpBA promoter of Pseudomonas aeruginosa by TrpI protein in vitro. 190 58

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
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PMID:E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription. 192 88

The promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli gene aroG (encoding the phenylalanine-sensitive 3-deoxy-arabinoheptulosonate-7-phosphate synthase) were located. Primer extension analysis and nuclease S1 mapping of in vivo transcripts were used to determine the 5' and 3' ends, respectively, of the mRNA. Both ends exhibited some heterogeneity with respect to length. The 3' end of the major molecular species was located within a region that has structural homology with known rho-independent terminators. The location of the aroG promoter was identified in both strands of the DNA by in vitro DNase I footprinting and methylation protection experiments with RNA polymerase. In these experiments, a region of up to 80 base pairs (bp) was protected by the binding of RNA polymerase. The location of the aroG operator was also identified in both strands of the DNA by in vitro DNase I footprinting with pure TyrR. TyrR protected 26 to 28 bp of DNA containing a 22-bp palindrome (TYR R box) and overlapping the -35 region of the promoter. Mutations in the aroG regulatory DNA were isolated by site-directed mutagenesis and cloned in a low-copy-number plasmid to generate aroG-lac fusions. The effects of the mutations on the regulation of aroG expression were determined by measuring the beta-galactosidase activities of the fusions in strains with tyrR, tyrR+, and multicopy tyrR+ genotypes. The results of this mutant analysis confirmed that the aroG operator contains a single TYR R box.
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PMID:Identification of the promoter, operator, and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG. 197 May 63

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions. The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate. The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation. We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.
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PMID:Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. 200 Mar 72

A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
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PMID:Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function. 204 69


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