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)

The spf gene of Escherichia coli encodes an unstable 109-nucleotide RNA, spot 42 RNA; the level of this RNA was reduced three- to fivefold when cells were grown in the presence of 3',5'-cyclic AMP (cAMP). We show that this regulation occurs through reduction in transcription and depends on both cAMP and the cAMP receptor protein (CRP) but is independent of the de novo protein synthesis. Through deletion analysis of the spf gene promoter, we have identified sequences that are important in the synthesis of spot 42 RNA. Deletion of sequences upstream of -77 completely eliminated the negative control of cAMP-CRP and resulted in high constitutive levels of transcription. This region contained a sequence that both conformed to the consensus binding site for cAMP-CRP in positively regulated promoters and acted as a cAMP-CRP binding site in a gel retardation assay. Deletion of sequences between positions -77 and -60 greatly reduced the level of transcription in the presence or absence of cAMP-CRP, indicating that at least part of this region is a binding site for a positive-acting transcription factor (or RNA polymerase itself). We propose that the proximity of the two sites defined here allows for the negative control of spf gene transcription by cAMP-CRP. In particular, if only one site at a time can be occupied, the binding of cAMP-CRP would interfere with the binding of a transcription factor.
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PMID:Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli. 245 12

The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations. We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor. Termination at all but the 421 position depends on rho factor. In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site. These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs. Several stop points also correspond to 3' end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly. Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.
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PMID:Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene. 247 37

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
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PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [alpha-33P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerase. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.
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PMID:Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius. 250 28

In vitro transcription by RNA polymerase II requires hydrolysis of the beta-gamma bond of ATP after the transcription complex forms, prior to RNA synthesis. It was observed that the presence of ATP during transcription complex formation inhibits subsequent transcription when the remaining 3 rNTPs are added. We now report that ATP or GTP inhibits transcription if either is present during transcription complex formation to added to preformed complexes. This inhibition is not due to purine rNTP degradation and occurs if as little as 2 mM ATP or 50 mM GTP is added to forming or preformed complexes. Deoxy derivatives of ATP inhibit similarly. AMP-PNP, a beta-gamma imido derivative, neither satisfies the energy requirement nor inhibits transcription if added to incubations of forming or of preformed transcription complexes.
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PMID:RNA polymerase II transcription complexes are destabilized by ATP or GTP. 276 22

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.
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PMID:Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase. 283 52

In a continuation of previous efforts to study the modified ATP requirements for RNA synthesis by poIR mutants of vesicular stomatitis virus (VSV), we have used a novel reconstitution assay to show that it is the template moiety of the mutants, not the polymerase proteins, which governs both the increased utilization of the ATP analog, beta, gamma-imido ATP (AMP-PMP), and the loss of a positive cooperativity-like response to varying ATP concentrations. Assays utilized uv-irradiated virus as a source of polymerase proteins and purified N-RNA as templates. Homologous and heterologous transcriptase reactions were carried out with wild-type (wt) virus and each of the two independently isolated poIR mutants. We show that in the presence of wt N-RNA template, substitution of AMP-PNP for ATP resulted in only approximately 5% of control RNA synthesis regardless of which source of polymerase was used. Furthermore, all reactions containing wt N-RNA template responded to varying ATP concentrations with a concave, upward-shaped Lineweaver-Burke plot generally indicative of positive cooperativity effects. In contrast, all reactions which utilized N-RNA templates from the poIR mutants showed an increased utilization of AMP-PNP (greater than 20%) and a more characteristic Michaelis-Menten response to changing ATP concentrations. These findings strongly support the notion that the template-associated nucleocapsid protein modulates the utilization of an ATP site which is directly or indirectly involved in VSV RNA synthesis.
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PMID:Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template. 284 22

Two overlapping promoters compete for RNA polymerase in the region that controls the expression of the galactose operon in Escherichia coli. Kinetics of open complex formation at P1 and P2 can be followed through the rate of formation of two specific abortive transcripts. The corresponding forward kinetic constants appear to be identical over a wide range of enzyme concentrations and temperatures, indicating that the two processes are strongly coupled. We propose a scheme accounting for our observations. In a first step, the competition between the two sites is a simple kinetic process, involving the "on" rate constants. In a second step, a slow reequilibration occurs, implicating the "off" rate constants and the conversion of one open complex to the other through a set of closed complexes. The first step is clearly affected when the complex between cyclic AMP and its receptor is bound at the activator site. An estimate of the various rate constants describing open complex formation at P1 and P2 is provided, as well as a qualitative description of the effect of the activator complex on these two pathways.
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PMID:Overlapping promoters and their control in Escherichia coli: the gal case. 301 Mar 19

The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo.
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PMID:Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site. 301 84

The molecular mechanisms whereby RNA polymerase, catabolite activator protein (CAP), and cyclic AMP (cAMP) participate in transcriptional regulation at the galactose operon have been probed by a variety of in vitro techniques. Interactions between purified proteins and promoter-containing DNA fragments were assayed by gel electrophoresis, by resistance to restriction endonuclease digestion, and by monitoring runoff transcripts. The data bear on the multiple functions that CAP performs in gal control. A CAP-cAMP complex can exclude RNA polymerase from one of the two overlapping promoter regions (P2), thereby targeting the enzyme to the other (P1); this process is markedly influenced by the cAMP level. In addition, a second CAP molecule is involved in a cooperative process, which, at low cAMP, is required for efficient formation of transcriptionally competent complexes at P1. This second CAP may serve to stabilize the 1:1:1 CAP-polymerase-gal DNA intermediate under physiological conditions, thus enhancing initiation from P1 relative to P2. Kinetic analysis reveals that the modest effect of CAP on the rate of P1 open complex formation can be resolved into about a 4-fold increase in the binding of RNA polymerase to the P1 region, plus a 1.5-fold elevation in the rate of isomerization of enzyme-promoter complexes to the open state.
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PMID:Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli. 302 95


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