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)

RNA polymerase II-specific transcription requires, in addition to auxiliary protein factors, the hydrolysis of the beta-gamma phosphate bond of ATP. The nonhydrolyzable analog of ATP, imidoadenosine triphosphate does not suffice for specific in vitro transcription (Bunick, D., Zandomeni, R., Ackerman, S., and Weinmann, R. (1982) Cell 29, 877-886), although it can be incorporated into RNA. The experiments presented here suggest two energy-dependent steps in RNA polymerase II transcription. One of these steps is required at, or close to, the point of initiation, as determined by 5' end primer extension analysis. In vitro transcription occurs efficiently in vitro when imidoadenosine triphosphate is supplemented with dATP to fulfill the energy requirement. In the presence both of imidoadenosine triphosphate and imidoguanosine triphosphate, the concentration of dATP required for transcription initiation is dramatically increased. This suggests that ATP and GTP are co-substrates in transcription initiation, supporting the role of protein kinase II in this process (Zandomeni, R., Zandomeni, M. C., Shugar, D., and Weinmann, R. (1986) J. Biol. Chem. 261, 3414-3419). The concentration of dATP required for maximal initiation is inadequate for the production of full-length transcripts, suggesting a second energy-dependent step in the RNA elongation process. Since the elongation step is unaffected by the presence of imidoguanosine triphosphate, GTP beta-gamma phosphate bond hydrolysis appears to be required only for initiation.
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PMID:Purine triphosphate beta-gamma bond hydrolysis requirements for RNA polymerase II transcription initiation and elongation. 369 65

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.
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PMID:Purification and characterization of two new high molecular weight forms of DNA polymerase delta. 395 90

A DNA primase activity has been purified from the budding yeast Saccharomyces. The resulting preparation was nearly homogeneous and was devoid of DNA and RNA polymerase activities. The primase activity cofractionated with a Mr 65,000 polypeptide in sedimentation and chromatography procedures, and the native molecular weight of the enzyme corresponded closely to this value suggesting that the primase or an active proteolytic fragment of the protein exists as a monomer. Both heat-denatured calf thymus DNA and poly(dT) could be utilized by the enzyme as templates. Primase exhibited an absolute requirement for divalent cations and for rATP on a poly(dT) template. Although it required the ribonucleotide to initiate primer chains, the enzyme could incorporate the deoxynucleotide into primers. The product of the primase-catalyzed reaction was an oligonucleotide of discrete length (11-13 nucleotides), and oligonucleotides that were apparently dimers of this unit length were also observed. Primers that were synthesized were virtually identical in size in both the presence and absence of dATP incorporation. Although the bulk of DNA primase activity was isolated as a "free" enzyme, a portion of cellular primase activity co-chromatographed with DNA polymerase suggesting an association between these enzymes similar to that found in several higher eukaryotes.
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PMID:A DNA primase from yeast. Purification and partial characterization. 398 42

Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT). Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts. The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase. Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis. The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur. During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway. The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme. Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA. However the ATP analogue cannot be incorporated into longer products than a trinucleotide. On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis. Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.
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PMID:Poly(dAT) dependent trinucleotide synthesis catalysed by wheat germ RNA polymerase II. Effects of nucleotide substrates and cordycepin triphosphate. 404 41

RNA polymerase has been purified from vegetative cells of Bacillus brevis and resolved into "core" enzyme and sigma factor. The purified enzyme is rapidly inactivated by incubation at low temperatures in the presence of 1-2 mM ATP, dATP, or NAD(+), while other nucleotides at this concentration have little or no effect. Inactivation is not accompanied by the incorporation of an adenylyl or phosphoryl moiety into RNA polymerase; nevertheless, it is essentially irreversible. DNA, high concentrations of glycerol, as well as low concentrations (1 mM) of orthophosphate protect RNA polymerase from the nucleotide-dependent inactivation.A similar inactivation of RNA polymerase in the presence of ATP is observed with crude preparations from Bacillus subtilis and Bacillus polymyxa. This phenomenon may represent a novel mode of regulation of transcription that does not involve a covalent modification of RNA polymerase or its interaction with other protein factors, but rather is due to a structural transition to an inactive form induced by small molecules.
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PMID:Nucleotide-dependent inactivation of RNA polymerase from Bacillus brevis. 440 27

The in vitro conversion of single-stranded DNA from bacteriophage fd to duplex structures depends on E. coli RNA polymerase, DNA polymerase III, riboand deoxyribonucleoside triphosphates, Mg(+2), spermidine or DNA-unwinding protein of E. coli, and two additional protein factors, referred to here as Factors I and II. These two factors are also essential for dTMP incorporation catalyzed by DNA polymerase III and dependent on poly(dA).oligo(dT) primer-template. In the latter reaction, there is an absolute dependency on ATP or dATP.
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PMID:Involvement of two protein factors and ATP in in vitro DNA synthesis catalyzed by DNA polymerase 3 of Escherichia coli. 458 95

Protein factor rho catalyzes site-specific termination of transcription in a reaction requiring hydrolysis of nucleoside triphosphate with eventual release of RNA from RNA polymerase and DNA template. We have characterized the rho-catalyzed RNA release reaction using isolated transcription complexes. Transcription complexes containing T7 D111 DNA, RNA polymerase, and 3H-labeled nascent RNA were formed and isolated by gel filtration on an Agarose 5M column. When the ternary complexes were incubated with rho factor in the presence of ATP, or dATP, significant amounts of nascet RNA were released from the complexes as determined in a membrane filtration assay. Gel electrophoretic analysis of RNA has revealed that rho releases selected species of discrete-sized RNA from among those originally present in the ternary complexes. These results show that rho essentially acts to release RNA from those ternary complexes which have come to pause, and that this reaction proceeds in a discrete step separately from the pausing of RNA synthesis. Under the conditions used, the extent of RNA release widely varied at individual pausing sites and thus the action of rho exhibited certain site-selectivity.
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PMID:Studies of RNA release reaction catalyzed by E. coli transcription termination factor rho using isolated ternary transcription complexes. 616 Apr 71

DNA polymerase from Micrococcus luteus and RNA polymerase from E. coli catalyze the synthesis of poly(dA) with poly(dT) template, in the presence of ATP and [alpha-32P]dATP. The reaction is completely dependent on poly(A) primer synthesis. Poly(A) chains are covalently extended by DNA polymerase. Primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA). The length of RNA and DNA products appears to be relatively variable. The size of the DNA is less than 3 000 nucleotides.
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PMID:Ribonuclease H from chick embryos cleaves precisely at the junction between the RNA and DNA portion of the hybrid helix. 618 57

The 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl) nucleoside 5'-triphosphate derivatives of adenine (3'-azido, x-dATP) and guanine (3'-azido, x-dGTP) were prepared by chemical phosphorylation of the corresponding nucleosides. The compounds were characterized by 31P and 1H NMR, high performance liquid chromatography, IR, and TLC. The compounds were examined kinetically and observed to be linear mixed inhibitors for the DNA-dependent RNA polymerase of Escherichia coli (EC 2.7.7.6); Ki values for the 3'-azido, x-dATP and 3'-azido, x-dGTP compounds are 33 and 0.95 microM, respectively. Neither compound functions as an alternate substrate or as a chain terminator during the normal kinetic time course. The 3'-azido, x-dGTP does exhibit a slow time-dependent irreversible inhibition and may therefore function as an alternate substrate and chain terminator with prolonged incubation. Both compounds (3'-azido, x-dATP and 3'-azido, x-dGTP) are photolabile and will derivatize lysine in a coupled photolytic reaction.
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PMID:RNA polymerase. Synthesis and kinetic inhibition by 9-(3'-azido-3'-deoxy-beta-D-xylofuranosyl) derivatives of 5'-ATP and 5'-GTP. 654 40

The energy requirement for specific transcription initiation and elongation by the human RNA polymerase II system was studied in vitro using partially purified transcription factors from HeLa cell nuclear extracts. The synthesis of the 536-nucleotide long run-off transcript resulting from initiation at the adenovirus major late promoter was found to be dependent upon the presence of either ATP or dATP (with the imido derivative adenyl-5'-yl imidodiphosphate being used as the substrate for the RNA polymerase elongation reaction). An identical requirement for hydrolysis of the phosphate bond in an adenosine nucleotide was observed for the synthesis of the decanucleotide transcribed from the major late promoter in the absence of the GTP substrate. In contrast, the nonhydrolyzable analog adenyl-5'-yl imidodiphosphate fully substitutes for ATP during the subsequent elongation of these short transcripts, which demonstrates that the energy requirement occurs at an earlier step of the transcription reaction. Thus the particular transcription factor that requires ATP (or dATP) hydrolysis for its function must act prior to, or concomitant with, formation of the first few phosphodiester linkages by the RNA polymerase II.
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PMID:Energy requirement for specific transcription initiation by the human RNA polymerase II system. 671 44


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