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 phenotypes of 24 mutants that successively delete DNA sequences adjacent to the 5' end of the Saccharomyces cerevisiae (yeast) his3 structural gene are described. Deletions retaining greater than 155 base pairs before the mRNA coding sequences are phenotypically indistinguishable from the wild-type his3 allele. Deletions having end points between 113 and 65 base pairs before the transcription initiation site express his3 at reduced levels. Mutations retaining less than 45 base pairs are indistinguishable from null alleles of the his3 locus. These results indicate (i) that a sequence(s) located 113--155 base pairs upstream from the transcribed region is necessary for wild-type expression and (ii) that the T-A-T-A box (a sequence in front of most eukaryotic genes) is not sufficient for wild-type promoter function. Thus, the yeast his3 promoter region appears large when compared with prokaryotic promoters, suggesting that it may be more complex than a simple site of interaction between RNA polymerase and DNA.
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PMID:Deletion mapping a eukaryotic promoter. 702 62

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.
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PMID:Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli. 702 6

An average of 0.44 molecule each of the initiation factor sigma and the RNA polymerase binding protein y and 0.54 molecule of the subunit gamma per molecule of Lactobacillus curvatus DNA-dependent RNA polymerase have been found in the cell. Free factor y displaces sigma from free holo enzyme, E sigma. The formation of a binary complex from Ey, free sigma, and poly[d(A-T)], leads to immediate release of factor y. The release of the sigma factor occurs upon the transition of the binary to a ternary complex. A mixture of E and sigma forms binary complexes with all T7 DNA HpaII restriction fragments. In contrast a mixture of Ey and sigma binds selectively to promoter-containing DNA fragments, indicating that the stimulatory effect of y on transcription is due to an increase in the rate of promoter selection. The same RNA products are synthesised by E sigma and by Ey plus sigma with T7 DNA as template. Thus the nonspecific complexes formed by E sigma and T7 DNA are nonproductive. On the basis of these findings we propose a model for the transcription cycle in Lactobacillus curvatus.
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PMID:The role of the components sigma and y of the DNA-dependent RNA polymerase of Lactobacillus curvatus in promotor selection. 710 25

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.
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PMID:Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis. 711 96

RNA product distribution obtained during the transcription of poly[d(A-T)] by wheat germ RNA polymerase IIA under various experimental conditions was analyzed by high resolution polyacrylamide gel electrophoresis. Poly[r(A-U)] synthesis proceeded as if wheat germ RNA polymerase II was a non-processive enzyme: a ladder of RNA products of increasing lengths was obtained, which apparently, terminated at every other nucleotide. RNA release was not dependent upon nucleoside triphosphate substrate concentrations. A likely explanation would be that ternary complexes enzyme: DNA: RNA were very much unstable; moreover, oligonucleotides released were not re-used for further elongation by the enzyme.
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PMID:Non-processive transcription of poly[d(A-T)] by wheat germ RNA polymerase II. 716 Apr 87

Using a recombinant phage containing the mouse beta-Globin gene with lambda gtWES bacteriophage DNA, transcription initiation sites for Escherichia coli RNA polymerase and calf thymus RNA polymerase B were mapped at the 5' and 3' ends of the mouse beta-Globin gene. The bacterial enzyme was capable of initiating RNA synthesis at the 3' end site located at about 700 residues from the 3' end of the beta-Globin restriction enzyme map. Initiation at this site was more efficient than initiation at the known early lambda promotors (PL, PR). Calf thymus RNA polymerase B initiated transcription at the same sites as the bacterial enzyme but in this case maximum efficiency was at the 5' end site as compared to the 3' end site. Initiation of transcription occurs in the region of the d(T-A-T-A-A) sequence. Initiation efficiency at the 5' end site, as probed by the maximum rate of transcription, was shown to depend partly upon the presence of the adjacent sequences upstream and downstream of the 5' initiation site.
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PMID:Identification of transcription initiation sites for bacterial RNA polymerase and eukaryotic RNA polymerase B on the 5' end of the mouse beta-Globin gene. 728 30

Incubation of RNA polymerase with poly[d(A-T)n] template results in a binary enzyme-DNA complex. Further addition of the dinucleotide UpA and [alpha-32P]UTP results in catalytic formation of the labeled trinucleotide UpApU until substrate exhaustion. In contrast, incubation of binary enzyme-DNA complexes with ApU and [alpha-32P]ATP results in labeled ApUpA formation to an extent that is stoichiometric with the amount of enzyme present despite an excess of substrates. The occurrence of ApUpA in a stable DNA-enzyme-RNA ternary complex is shown by gel exclusion chromatography, Millipore filtration, and the ability of ternary complexes to support subsequent RNA chain elongation. Radioactivity is not bound to Millipore filters when purified, labeled ApUpA is added to enzyme-DNA binary complexes. Hence, phosphodiester bond formation is required for stable ternary complex formation. The absence of the delta subunit of RNA polymerase or the addition of rifampicin to the reaction before ribonucleotide substrates results in catalytic ApUpA formation instead of stable ternary complexes.
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PMID:Stable RNA-DNA-RNA polymerase complexes can accompany formation of a single phosphodiester bond. 737 Feb 24

The template specificity of DNA-dependent RNA polymerases I and II (ribonucleoside 5'-triphosphate : RNA nucleotidyltransferase [EC 2.7.7.6]) of Dictyostelium discoideum was investigated with several synthetic polynucleotides at three different stages of development. Both the enzymes exhibited several common characteristics for some templates, and distinctly different properties for other ones. Of single-stranded homopolymers, the strands of pyrimidine nucleotides were much transcribed in the order of poly(dC) greater than poly(dT). The double-stranded homopolymers, poly(dA). poly(dT)) and poly(dG).poly(dC) were transcribed asymmetrically, the pyrimidine-containing strand being preferentially read. Transcription of double-stranded alternating copolymers, poly([d(A-T)].poly[d(A-T)] and poly[d(G-C)].poly[d(g-C)] occurred to some extent. Except for poly(rC), all of the single-stranded ribonucleotide homopolymers were extremely poor as templates. The polynucleotides containing thymidine were more efficient templates for polymerase I than polymerase II. The enzyme activities of the two polymerases were more or less variable with some polynucleotides among three stages of development, suggesting the possibility that D. discoideum RNA polymerases tend to change their template specificity during development.
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PMID:Template specificity of DNA-dependent RNA polymerase I and II for synthetic polynucleotides during development of the cellular slime mold Dictyostelium discoideum. 739 Sep 95

Using mild sonication, nucleoplasmic, nucleolar, and subnucleolar P-3 and S-3 chromatin fractions are isolated from rat liver nuclei. These fractions differ widely (over 80-fold) from each other in transcriptional activity as measured by the chromatin bound engaged RNA polymerases. Chemical analyses indicate that the active chromatin, e.g. P-3 and nucleolar fractions, are rich in RNA and protein as compared to the inactive chromatin, e.g. nucleoplasmic, and S-3 fractions. However, the DNA base content are all the same, showing 40% GC and 60% AT, including P-3 which is enriched in rDNA. Polyacrylamide gel electrophoresis of the 0.25 N HCl extracted proteins shows that all five histones are present in active chromatin. Additionally, the gel reveals two protein bands, one ahead of histone H2B and another ahead of histone H4, that are diminished or missing from the inactive chromatin. On the other hand, there is a fast moving protein band ahead of H4 in the inactive chromatin that is almost absent in the active chromatin. Transcriptional tests using E. coli RNA polymerase and several synthetic DNA templates of known base content and sequence indicate that the 0.25 N HCl soluble protein extracts from active chromatin contain activator proteins which are capable of countering the histone suppressors present in the extracts in a DNA base and sequence specific manner. The data show that although the histone suppressors are able to strongly inhibit the template function of poly[d(A-T)], the protein activators are able to overcome the suppressor activity and stimulate RNA synthesis several-fold when poly(dA).poly(dT) or poly(dT) is used.
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PMID:Studies on the isolated transcriptionally active and inactive chromatin fractions from rat liver nuclei. 760 68

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.
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PMID:Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase. 773 58


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