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)

Hg-UMP-containing transcripts made from chick erythroid chromatins with E. coli RNA polymerase hybridize to chick globin cDNA. Contamination with endogenous globin RNA has been largely removed by purification on SH-agarose columns at 55 degrees C. Some endogenous globin mRNA sequences remain, probably as hybrids with "anti-sense" Hg-transcripts produced by RNA-dependent RNA synthesis. Heating to 115 degrees C before SH-agarose chromatography eliminates these contaminants. Hg-transcripts from adult and embryonic erythroid chromatins purified by this method are hybridized to globin cDNA; they contain a 4- to 6-fold higher proportion of globin-specific sequences (10-13 PPM) than do transcripts from brain chromatin. Dissociation of erythroid chromatins in salt and urea, followed by reconstitution using standard methods, destroys even this low degree of specificity.
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PMID:Is there specific transcription from isolated chromatin? 65 20

The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.
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PMID:The role of FIS in trans activation of stable RNA operons of E. coli. 169 Jan 24

We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.
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PMID:Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor. 267 69

The developmental regulation of two kinds of Xenopus 5S RNA genes (oocyte and somatic types) can be explained by differences in the stability of protein-protein and protein-DNA interactions in a transcription complex that directs transcription initiation by RNA polymerase III. Dissociation of transcription factors from oocyte 5S RNA genes during development allows them to be repressed by chromatin assembly. In the same cells, somatic 5S RNA genes remain active because their transcription complexes are stable.
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PMID:Developmental regulation of two 5S ribosomal RNA genes. 342 Apr 14

Dissociation of RNA and DNA from Escherichia coli RNA polymerase in transcription complexes prepared with enzyme molecules located within and near a rho-dependent transcription termination region on bacteriophage T7 D111 DNA has been studied using a membrane filter-binding assay. Rho protein with ATP present mediated rapid (half-time approximately 27 s) simultaneous dissociation of about 50% of both RNA and DNA. RNA molecules were preferentially released from enzyme molecules located within the termination region. Rapid release of RNA and DNA depended on a nucleoside triphosphate but did not depend on sigma factor. Pretreatment of complexes with ribonuclease prevented dissociation of DNA. Nearly simultaneous dissociation of both RNA and DNA was also detected after a lag of 3 min when the isolated transcription complexes were incubated with all four ribonucleoside triphosphates in the absence of rho factor. In this case, release presumably occurred at the rho-independent termination site that is 5990 nucleotides downstream from the A1 promoter. Thus, the dissociation of DNA from RNA polymerase at rho-dependent and rho-independent transcription termination sites is coupled with or occurs spontaneously soon after the release of transcripts at both sites.
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PMID:Transcription termination factor rho mediates simultaneous release of RNA transcripts and DNA template from complexes with Escherichia coli RNA polymerase. 388 62

A gene-specific transcription factor, called USF, has been partially purified from HeLa cell nuclear extracts. Addition of USF results in a 10 to 20 fold increase in transcription from the adenovirus major late promoter in an in vitro system reconstituted with transcription factors TFIIB, TFIID, TFIIE, and RNA polymerase II. Binding of USF to the promoter inhibits DNAase I cleavages over a 20 base pair region just upstream of the -45 to +35 region shown previously to interact with TFIID. More discriminating footprint analyses using methidiumpropyl-EDTA-Fe(II) as the cleaving agent indicate that USF interacts primarily with the small palindromic DNA sequence GGCCACGTGACC located between positions -63 and -52 of the major late promoter, while TFIID interacts primarily with a 10 base pair DNA region centered on the consensus TATA sequence. Dissociation rate measurements indicate a cooperative interaction between USF and TFIID when simultaneously bound to the promoter DNA.
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PMID:Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. 407 92

A deoxynucleoprotein complex (DNP-1) isolated from simian virus 40 (SV40) after disruption of the virus in an alkaline buffer contains the viral deoxyribonucleic acid (DNA) and four minor structural polypeptides. Dissociation of DNP-I by equilibrium centrifugation in CsCl yields a complex (DNP-II) that contains a small amount of polypeptide tightly bound to the viral DNA. Studies of the template activity of these deoxynucleoprotein complexes in vitro with Escherichia coli transcriptase show that the rate of transcription of DNP-I and DNP-II is 30 and 80%, respectively, compared with that of deproteinized SV40 DNA component I. In dimethyl sulfoxide gradients, the complementary ribonucleic acid (cRNA) synthesized from DNP-I is one-third to one-half the size of the cRNA species from DNA-I and DNP-II. Competition hybridization experiments show that with the E. coli transcriptase only a portion (about one-half) of the SV40 genome is transcribed with DNP-I as template, whereas most or all of the genome is transcribed with DNP-II as template. The template activity of the deoxynucleoprotein complexes with a highly active form II ribonucleic acid polymerase prepared from SV40-infected permissive cells follows similar transcription kinetics. The results indicate that structural nucleoproteins of SV40 bind nonrandomly to the viral DNA and effect the transcription of some subset of its sequences in vitro.
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PMID:Structure and function of the polypeptides in simian virus 40. II. Transcription of subviral deoxynucleoprotein complexes in vitro. 433 39

The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polymerase holoenzyme (RNAP) and the lambda PR promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 degrees C, 37 degrees C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at PR by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction. The association reaction between RNAP and lambda PR was investigated under ionic conditions where the process is essentially irreversible, and under pseudo first-order conditions of excess polymerase. The pseudo first-order rate constant is directly proportional to the concentration of active polymerase over the entire range investigated (2 to 10 nM) at both 25 degrees C and 37 degrees C, within experimental uncertainty. Second-order association rate constants (ka), calculated from these data at standard ionic conditions (0.12 M-KCl, 0.01 M-MgCl2, 0.04 M-Tris (pH 8)), were strongly temperature-dependent: ka = (2.6 +/- 0.4) X 10(6) M-1 S-1 at 37 degrees C and ka = (7.2 +/- 1.4) X 10(5) M-1 s-1 at 25 degrees C, corresponding to an activation energy of the association reaction of approximately 20 +/- 5 kcal. In addition, ka decreases strongly with increasing KCl concentration, corresponding to the net release of the thermodynamic equivalent of at least nine monovalent ions prior to or during the rate-limiting step of the association reaction. This strong dependence of ka on the ionic environment suggests that inorganic cations should be considered as possible regulators of in vivo transcription initiation. Dissociation rate constants (kd) were also measured under irreversible reaction conditions. At the standard ionic conditions, kd = (2.2 +/- 0.3) X 10(-5) s-1 at 37 degrees C and kd = (4.0 +/- 0.4) X 10(-5) s-1 at 25 degrees C. The increase in kd with decreasing temperature corresponds to a negative activation energy of dissociation (-9 +/- 4 kcal). In addition, kd increases with increasing KCl concentration, corresponding to the net uptake of the thermodynamic equivalent of at least six monovalent ions in or prior to the rate-limiting step of the dissociation reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter. 623 75

E. coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+. Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis. The reaction was realized in the absence of added nucleoside triphosphates. Nucleoside triphosphates are low molecular mass products of the reaction. The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration. The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis. Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture. RNA as short as 70-8 nucleotides long can be produced at the conditions used. It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA. Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation. The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised.
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PMID:[Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase]. 626 61

The leftmost portion of the coliphage T7 genome is transcribed by the RNA polymerase Escherichia coli immediately after infection. This "early" operon is delineated by three promoters on the left, and a transcriptional terminator on the right. The terminator is efficient both in vivo, and with highly purified RNA polymerase in vitro. We have studied termination in vitro, using synchronously initiated transcription reactions with low enzyme:DNA ratios. We show that recognition of the stop signal and release of RNA product are relatively rapid. Dissociation of the enzyme from the DNA is quite slow, and probably rate-limiting for re-cycling of the polymerase. It is well established that the sigma subunit of RNA polymerase is required for specific initiation, but redundant during RNA elongation. By exploiting antisigma antiserum we have obtained evidence that sigma is also redundant during all steps of termination in vitro.
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PMID:Termination of transcription of the coliphage T7 "early" operon in vitro: slowness of enzyme release, and lack of any role for sigma. 679 94


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