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)

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.
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PMID:Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland. 122 Jun 81

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.
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PMID:Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos. 124 20

DNA-dependent RNA polymerase from Escherchia coli was used to transcribe chromatin from human leukocytes and purified human DNA. RNA was labeled at the 5' terminus with either [gamma-32P]ATP or [gamma-32P]GTP and internally with [3H]UTP. Determination of the average chain length of the RNA molecules by the ratio of moles of 3H-labeled nucleotide incorporated to moles 32P-labeled nucleotide incorporated showed that the size of the transcript of purified DNA was about 2 1/2 times greater than those from chromatin. The percentage of chains initiated with ATP and GTP was observed to vary with the template, the ATP to GTP ratio being greater on chromatin. The kinetics of 3H and 32P hybridization of transcripts of purified DNA showed hybridization primarily to nonrepetitive sequences. Transcripts from the chromatin templates when hybridized to DNA showed a larger proportion of RNase resistance of the 32P-termini at low Cot's.
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PMID:Template restriction in human chromatin. 126 Aug 58

A photoactive nucleotide analogue of UTP, 5-azido-uridine 5'-triphosphate (5-N3UTP), has been demonstrated to interact with the RNA polymerase of the vesicular stomatitis virus (VSV) transcription complex. Kinetic studies indicated that 5-N3UTP served as an efficient replacement for UTP in in vitro polymerase reactions. The Km for the azido analogue was 27 microM and that of the natural substrate, UTP, was 7 microM. Photolysis of [gamma-32P]5-N3UTP in the presence of VSV transcription complexes resulted in selective radio-labelling of the L protein. This photolabelling was saturable with an apparent Kd of 28 microM. The L protein was protected from [gamma-32P]5-N3UTP-mediated photolabelling by competing natural substrates (UTP, CTP, ATP, GTP). The stoichiometry of photoprobe incorporation into the transcription complex was close to unity with respect to the L protein. These data provide evidence that the nucleotide-binding domain of the VSV RNA polymerase contains amino acid residues of the L protein.
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PMID:The L protein of vesicular stomatitis virus transcription complexes is specifically photolabelled by 5-azido-uridine 5'-triphosphate, an analogue of the RNA polymerase substrate uridine 5'-triphosphate. 130 62

A photoaffinity analogue of ATP, 8-azido-adenosine 5'-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 microM-8N3ATP and 1.3 at 100 microM-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.
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PMID:Identification and characterization of serine/threonine protein kinase activity intrinsic to the L protein of vesicular stomatitis virus New Jersey. 130 63

p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl-terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation-stimulated (PCS/type-2A) protein phosphatase and the active catalytic subunit of the ATP,Mg-dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10-20-fold-better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100-fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72-kDa, 65-kDa and 55-kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8-fold) by the PCSH1 phosphatase and is about a 40-fold and 95-fold-better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl-terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km = 10 microM, Vmax = 3882 nmol.min-1.mg-1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor-1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.
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PMID:Specificity of the polycation-stimulated (type-2A) and ATP,Mg-dependent (type-1) protein phosphatases toward substrates phosphorylated by P34cdc2 kinase. 131 64

Mammalian RNA polymerase II contains at the C terminus of its largest subunit an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The phosphorylation of this domain is thought to play an important role in the transition of RNA polymerase II from a preinitiation complex to an elongating complex. The unphosphorylated form of RNA polymerase II is designated IIA, whereas the phosphorylated form is designated IIO. In an effort to determine the consequence of C-terminal domain phosphorylation on complex formation, 32P-labeled RNA polymerases IIA and IIO were prepared and examined for their ability to form a stable preinitiation complex on the adenovirus-2 major late promoter in the presence of a reconstituted HeLa cell transcription extract. Preinitiation complexes were formed in the absence of ATP and purified from free RNA polymerase II by chromatography on Sepharose CL-4B. The state of phosphorylation of the largest subunit was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the transcriptional activity was determined by assaying specific transcript formation upon the addition of nucleotides and a competing DNA template. RNA polymerase IIA was recovered in transcriptionally active complexes in reactions in which the input enzyme was RNA polymerase IIA. In reactions with RNA polymerase IIO as the input enzyme, no IIO was recovered in excluded fractions that normally contain preinitiation complex. In reactions with equimolar amounts of RNA polymerases IIO and IIA, purified preinitiation complexes contained almost exclusively RNA polymerase HA. These results support the idea that RNA polymerase II containing an unphosphorylated C-terminal domain preferentially associates with the adenovirus-2 major late promoter. The state of phosphorylation of the C-terminal domain can, therefore, directly influence preinitiation complex formation. We also report here the presence of an activity in HeLa cell extracts that catalyzes dephosphorylation of the C-terminal domain, thereby converting RNA polymerase IIO to IIA. This C-terminal domain phosphatase is specific in that it does not catalyze the dephosphorylation of a serine residue phosphorylated by casein kinase II. The presence of a C-terminal domain phosphatase in in vitro transcription reactions containing RNA polymerase IIO results in the formation of RNA polymerase IIA. This RNA polymerase IIA associates preferentially with preinitiation complexes.
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PMID:The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. 131 3

In order to understand translocation in transcription, it is important to develop a continuous functional assay for RNA polymerase (RNAP) activity in vitro. Fluorescent derivatives of ATP, UTP, UpA, and CpA with aminonaphthalene-5-sulfonic acid (AmNS) attached to the nucleotide triphosphates via a gamma-phosphoramidate bond or to the dinucleotide monophosphates via a 5'-secondary amine linkage were synthesized [Tyagi, S.C., & Wu, F.Y.-H. (1987) J. Biol. Chem. 262, 10684-10688]. The fluorescent emission spectra of (5'-AmNS)UpA, (5'-AmNS)CpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP overlap the absorption spectrum of co-substituted RNA polymerase (Co-RNAP) and ensure fluorescence resonance energy transfer (FRET) between the fluorescent analog and Co(II) in Co-RNAP. The binding constants at a single site for (gamma-AmNS)ATP, (gamma-AmNS)UTP, (5'-AmNS)UpA, and (5'-AmNS)CpA were observed to be 7.11, 5.26, 0.52, and 0.61 microM, respectively, in Co-RNAP and 5.70, 3.42, 0.12, and 0.21 microM, respectively, in Zn-RNAP. (8-AmTEMPO)ATP, with the spin probe AmTEMPO attached to the C-8 position at ATP [Tyagi, S.C. (1991) J. Biol. Chem. 266, 17936-17940], and Mn(3'-OCH3)UTP were synthesized. Mn-(II)-substituted RNA polymerase (Mn-RNAP) is prepared. The single site binding constants for (8-Am-TEMPO)ATP and Mn(3'-OCH3)UTP were 3.58 and 2.35 microM in Zn-RNAP and 5.77 and 3.43 microM in Mn-RNAP, respectively. These results indicate that dinucleotides bind much more tightly than mononucleotides to RNAP and that the binding constants are roughly the same for both Co- and Zn-substituted RNAP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli. 132 60

We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway.
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PMID:Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis. 137 99

We previously purified RNA polymerase II transcription factor delta from rat liver and found that it has an associated DNA-dependent ATPase (dATPase) activity. In this report, we show that delta is also closely associated with a protein kinase activity that catalyzes phosphorylation of the largest subunit of RNA polymerase II. Kinase activity copurifies with transcription and DNA-dependent ATPase (dATPase) activities when delta is analyzed by anion- and cation-exchange HPLC as well as by sucrose gradient sedimentation, arguing that delta possesses all three activities. Phosphorylation of the largest subunits of both rat and yeast RNA polymerase II is stimulated by DNA, whereas phosphorylation of a synthetic peptide containing multiple copies of the carboxyl-terminal heptapeptide repeat is not. Although both ATP and GTP appear to function as phosphate donors, GTP is utilized less than 10% as well as ATP. These findings suggest that delta may exert its action in transcription at least in part through a mechanism involving phosphorylation of the largest subunit of RNA polymerase II.
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PMID:A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. 138 28


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