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)

We have prepared RNA polymerase II preinitiation complexes by incubating templates containing the adenovirus 2 major late promoter with HeLa cell nuclear extracts in the absence of nucleoside triphosphates. These preinitiation complexes are partially purified by gel filtration and are then provided with the appropriate substrates to allow either one or two phosphodiester bonds to be formed. When substrates that allow only one bond to form are used, no stable ternary complex is obtained and no RNA is made that can be incorporated into longer RNA chains. A stable complex is obtained, however, if the RNA polymerase can make two bonds. The production of a stable ternary complex requires ATP or dATP and is inhibited by alpha-amanitin. In the course of exploring the energy requirement for initiation we found that dATP may be incorporated, in the absence of ATP, as the initial base of the RNA. However, deoxyribonucleotides are not appreciably incorporated into the body of the transcript after the first two bases have been added to the growing chain.
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PMID:Transcription initiation by RNA polymerase II in vitro. At least two nucleotides must be added to form a stable ternary complex. 243 60

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

We have investigated the role that ATP plays in the synthesis of accurately initiated transcripts from the adenovirus 2 major late and mouse interleukin-3 promoters by a purified RNA polymerase II transcription system prepared from rat liver. The synthesis of 250-330 nucleotide run-off transcripts and 4-9 nucleotide Sarkosyl-resistant transcription intermediates requires ATP both for RNA synthesis and for activation of the system prior to RNA synthesis. Activation specifically requires an adenine nucleoside triphosphate containing a hydrolyzable beta, gamma-phosphoanhydride bond. ATP, adenine-9-beta-D-arabinofuranoside (araATP), and dATP are potent activators of transcription; they activate transcription to 50% of maximum at 2 microM. ATP analogs containing nonhydrolyzable beta, gamma-phosphoanhydride bonds such as adenyl-5'-yl imidodephosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and adenosine 5'-O-(thio)triphosphate (ATP gamma S) function efficiently in chain elongation, but do not activate transcription. Furthermore, ATP gamma S is a potent, reversible inhibitor of ATP activation. 20 microM ATP gamma S inhibits the synthesis of both full-length run-off transcripts and sarkosyl-resistant intermediates by 50% when the concentration of ATP is 10 microM. ATP gamma S inhibition can be overcome by high concentrations of ATP, dATP, araATP, or ddATP. Inhibition of the synthesis of Sarkosyl-resistant transcription intermediates by ATP gamma S is prevented by preincubation of the transcription enzymes and DNA template with ATP and magnesium prior to the addition of ATP gamma S and the remaining ribonucleoside triphosphates. Thus we argue that ATP activates the transcription system in a step prior to RNA synthesis.
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PMID:ATP activates transcription initiation from promoters by RNA polymerase II in a reversible step prior to RNA synthesis. 244 31

DNA fragments containing the adenovirus 2 major late or simian virus 40 early promoters were attached to a solid support via a biotin-streptavidin linkage at one end of the fragment, upstream of the RNA start site. Templates immobilized in this manner were incubated with HeLa cell nuclear extracts to form preinitiation complexes containing RNA polymerase II and accessory proteins required for faithful in vitro transcription. These complexes did not require ATP or dATP for assembly, were sensitive to 0.25% Sarkosyl, and were stable to extensive washing. Their incubation with specific combinations of nucleoside triphosphates resulted in the initiation of RNA chain polymerization in situ, while addition of the remaining nucleoside triphosphates was necessary to produce a full length runoff RNA. Transcriptional activity associated with preinitiation complexes was purified approximately 300-fold, relative to the unfractionated nuclear extract. The use of immobilized template permits considerable flexibility in experimental design, as substrates and inhibitors can be added and washed out of the reaction at each step. We exploited this property of the system to dissect the temporal substrate requirements for initiation of RNA synthesis. It is known from prior work that at least one step in the promoter-dependent RNA synthesis reaction requires an adenosine nucleotide that is hydrolyzable at the beta, gamma-position. This requirement is independent of the initiating nucleotide and can be satisfied by dATP, which is not ordinarily incorporated into the RNA product. We show here that the beta, gamma-hydrolyzable adenosine nucleotide must be present simultaneously with the initiating nucleoside triphosphates. No reaction occurred when complexes were incubated with dATP, washed to remove dATP, and incubated subsequently with the two initiating nucleotides.
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PMID:Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes. 246 95

Mammalian cells contain two forms of RNA polymerase II, designated IIO and IIA, that differ in the extent of phosphorylation within the C-terminal domain of their largest subunit. Phosphorylation of this domain, which results in the conversion of RNA polymerase IIA to IIO, may play an important role in the transition from the initiation to the elongation phase of transcription. A third form of the enzyme, RNA polymerase IIB, is found in vitro and lacks the repetitive C-terminal domain. Purified calf thymus RNA polymerase IIA was labeled selectively with casein kinase II in the presence of [gamma-32P]ATP and used as substrate for the identification and partial purification of factors that catalyze the conversion of RNA polymerase IIA to IIO. HeLa cell S-100 transcription extracts contain such an activity that cofractionates with factors essential for promoter-dependent transcription through heparin-Sepharose, DEAE-5PW, and DE52 chromatography. The activity is dependent on either ATP, GTP, or dATP, requires a hydrolyzable beta,gamma-phosphoanhydride bond, and cannot utilize pyrimidine nucleoside triphosphates. This observation supports the idea that the conversion activity is a protein kinase. Transcription of the major late promoter of adenovirus-2 was carried out in the presence of a reconstituted transcription extract containing purified RNA polymerases IIO, IIA, or IIB, and the nature of the elongating enzyme was determined by photoaffinity labeling. When the reaction was initiated with RNA polymerase IIO or IIB, nascent transcripts were found cross-linked to subunit IIo or IIb, respectively. However, when the reaction was initiated with RNA polymerase IIA, nascent transcripts were cross-linked to subunit IIo. Consequently, phosphorylation of the C-terminal domain of subunit IIa must have occurred prior to elongation. The copurification of RNA polymerase IIA to IIO conversion activity with factors essential for promoter-dependent transcription and the observation that RNA polymerase II containing an unphosphorylated C-terminal domain is phosphorylated prior to elongation suggest that protein kinases that phosphorylate the C-terminal domain of subunit IIa may play an essential role in transcription.
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PMID:The transition of RNA polymerase II from initiation to elongation is associated with phosphorylation of the carboxyl-terminal domain of subunit IIa. 258 85

A native gel electrophoresis DNA binding assay was used to resolve complexes formed on the adenovirus Major Late Promoter by general transcription factors and RNA polymerase II. Five sets of complexes containing distinct components were identified. These complexes were generated by sequential binding of TFIID, TFIIA, TFIIB, RNA polymerase II, and TFIIE. The relative positions of each of the factors in the complexes were determined by DNAase I footprint analysis. TFIIA, derived from yeast or mammalian cells, formed a complex with yeast TFIID and the TATA element. TFIIB bound to this complex and probably acts as a "bridge" to the polymerase and the initiation site. The addition of ATP or dATP, necessary for "activation" of transcription, resulted in an alteration of the footprint in the +20 to +30 region, the same area protected upon addition of TFIIE to the initiation complex. Addition of ribonucleotide triphosphates generated new complexes that contained accurately initiated transcripts associated with the transcription machinery and the template DNA. A model for the interactions of components in initiation of transcription by RNA polymerase II is proposed.
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PMID:Five intermediate complexes in transcription initiation by RNA polymerase II. 291 66

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

The purified RNA polymerase complex of vesicular stomatitis virus required added thiols for maximal activity, whereas polymerase activity from whole disrupted virions did not. Maximal activity of the purified polymerase complex required greater than or equal to 1 mM added dithiothreitol. The polymerase was inactivated by N-ethylmaleimide (NEM) at 0 degree C, with k2 = 528 +/- 26 M-1 min-1. Activity was recovered by addition of L protein, but not N or NS, to the NEM-inactivated complex, indicating that the NEM-sensitive group was present on the L protein. Nucleoside triphosphates protected the enzyme against inactivation by N-ethylmaleimide. ATP was most effective, with KD = 0.58 +/- 0.07 mM, a value close to the Km of ATP reported previously for initiation of RNA synthesis. dATP was nearly as effective, and GTP was slightly less effective than ATP. Non-hydrolyzable analogs of ATP protected weakly, whereas ADP and pyrimidine triphosphates gave very poor, but still measurable, protection. The ATP binding site thus identified differs from the protein kinase-associated ATP binding site identified on L protein by Sanchez et al. (Sanchez, A., De, B.P., and Banerjee, A. K. (1985) J. Gen. Virol. 66, 1025-1036) in having a substantially lower affinity for ATP. Two putative ATP binding sites were identified in the L protein amino acid sequence, but none were found in the N or NS sequences.
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PMID:Inactivation of the RNA polymerase of vesicular stomatitis virus by N-ethylmaleimide and protection by nucleoside triphosphates. Evidence for a second ATP binding site on L protein. 303 24

A kinetic study of productive RNA chain elongation indicates that adenosine 5'-[beta gamma-imido]triphosphate can serve as substrate in reactions catalysed by purified wheat-germ RNA polymerase II on a poly[d(A-T)] template. However, in contrast with the results obtained with the natural substrate ATP, the double-reciprocal plots, 1/velocity versus 1/[nucleotide], are not linear but characteristic of apparent negative co-operativity. The extent of the kinetic co-operativity is modified when the reactions are conducted in the additional presence of fixed amounts of an alternative substrate such as ATP or inhibitors such as dATP or cordycepin triphosphate. Analogous results are obtained whether the reactions are carried out in the presence of Mg2+ or Mn2+ as the metal ion cofactor. However, the data show that with Mn2+ the RNA polymerase is less specific in substrate recognition than with Mg2+. Tentative kinetic models are proposed to account for the rate measurements.
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PMID:Kinetic co-operativity of wheat-germ RNA polymerase II with adenosine 5'-[beta gamma-imido]triphosphate as substrate. 342 9

We have investigated the formation of the first phosphodiester bond by RNA polymerase II in vitro. The template was a cloned DNA bearing the adenovirus 2 major late promoter; transcription factors and RNA polymerase II were provided by a HeLa cell nuclear extract. Dinucleotide primers and single nucleoside triphosphates were used as substrates. We found that accurate initiation does occur when only one phosphodiester bond can be formed; however, all of the resulting dinucleotide-primed trimers are abortively initiated. Synthesis of the trimers by RNA polymerase II requires ATP or dATP and is sensitive to low concentrations of alpha-amanitin. Treatments which abolish the ability of the preinitiation complex to synthesize long RNAs also eliminate the ability to abortively initiate. Abortive initiation proceeds for at least one-half h at 25 degrees C, at which point up to 4 mol of transcript/mol of template have been synthesized. The level of abortive initiation (per template molecule) is not significantly reduced by 0.025% Sarkosyl or by 10-fold dilution of the reaction, consistent with the initiation complex remaining intact during abortive initiation.
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PMID:Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter. 366 20


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