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)

Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wehat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation. S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypepetide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.
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PMID:Methylation-dependent translation of viral messenger RNAs in vitro. 16 87

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (beta-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the beta-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing (32)P label only in the cap (m(7)G(32)pppm(6)A(m)-) were prepared by recapping beta-eliminated globin mRNAs with the vaccinia virus enzymes, [alpha-(32)P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a (32)P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.
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PMID:Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. 28 3

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.
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PMID:Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap. 44 45

HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.
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PMID:Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates. 62 55

In the presence of Mg(2+) and a specific dinucleotide primer (ApG or GpG), the influenza virion transcriptase synthesizes the eight discrete segments of complementary RNA (cRNA) containing polyadenylic acid (Plotch and Krug, J. Virol. 21:24-34, 1977). Virions were examined for their ability to cap and methylate cRNA containing di- or triphosphorylated 5' termini. By using the primers ppApG, pppApG, or ppGpG, viral cRNA was synthesized in vitro with [alpha-(32)P]-GTP and S-[methyl-(3)H]adenosylmethionine as labeled precursors. DEAE-Sephadex chromatography of the RNase T2 digest of the cRNA product demonstrated no (3)H incorporation at all and the absence of a (32)P-labeled cap structure. The 5' terminus of ppApG-primed cRNA could be capped and methylated by enzymes from vaccinia virus, indicating that the two 5'-terminal phosphates derived from the primer were preserved in the product cRNA. The cap structure formed by the vaccinia enzymes and released by RNase T2 digestion as m(7)GpppA(m)pGp was radioactively labeled at its 3'-terminal phosphate only when [alpha-(32)P]CTP was used as the labeled precursor during transcription. This indicates that the 5'-terminal sequence of the cRNA is ppApGpC and that, therefore, ppApG most probably initiates transcription exactly at the 3' GpCpU(OH) terminus of the virion RNA templates. Virions were also tested for their ability to cap and methylate ppApG in the absence of transcription. No such activities were detected, whereas under the same conditions the vaccinia virus enzymes successfully capped and methylated this compound. Consequently, these experiments, together with those reported earlier, have not detected in influenza virions any capping and methylating enzymes active on the 5'-initiated termini of viral cRNA chains synthesized in vitro, whether these termini possess one, two, or three phosphates. Some mechanism for capping and methylation of viral cRNA must, however, exist, because the viral mRNA (cRNA) synthesized in the infected cell contains 5'-terminal methylated cap structures (Krug et al., J. Virol. 20:45-53, 1976). Possible mechanisms are discussed.
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PMID:Absence of detectable capping and methylating enzymes in influenza virions. 70 57

Deletion and point mutants of T3 have been isolated and used to show that the early region of T3 DNA is organized in the same way as that of T7 DNA. Homologous early RNAs and proteins of the two phages have been identified by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Both phages have five early mRNA's, numbered 0.3, 0.7, 1,1.1 and 1.3 from left to right, although no T3 protein that corresponds to the 1.1 protein of T7 has yet been identified. In general, corresponding early RNAs and proteins of the two phages migrate differently on gels, indicating that they differ in molecular weight and/or conformation. In both T7 and T3, gene 0.3 is responsible for overcoming the DNA restriction system of the host, gene 0.7 specifies a protein kinase, gene 1 specifies a phage-specific RNA polymerase, and gene 1.3 specifies a polynucleotide ligase. The 0.3 protein of T3 is responsible for the S-adenosylmethionine cleaving activity (SAMase) induced after T3 (but not T7) infection. However, cleaving of S-adenosylmethionine does not appear to be the primary mechanism by which T3 overcomes host restriction, since at least one mutant of T3 has lost the SAMase activity without losing the ability to overcome host restriction.
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PMID:SAMase gene of bacteriophage T3 is responsible for overcoming host restriction. 78 4

Reovirus mRNA synthesized in vitro by the virus-associated RNA polymerase in the presence of S-adenosylmethionine contains blocked, methylated 5'-termini with the structure, m-7G(5')ppp(5')G-MpCp. The functional significance and possible mechanism of formation of this novel 5'-5' terminal nucleotide linkage are discussed.
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PMID:Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-. 105 11

Purified Newcastle disease virus contains an enzyme that incorporates the methyl group from S-adenosyl-L-methionine into RNA synthesized in vitro by the virion-associated RNA polymerase (RNA nucleotidyltransferase). Incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine was totally dependent upon RNA synthesis. The methylation reaction was completely inhibited by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-methionine to RNA products. Velocity sedimentation and hybridization of the in vitro product RNA indicated that both [3H]methyl and [32P]GMP labels resided in single-stranded 18S RNA molecules which were virus specific. Approximately 1 to 2 methyl groups were incorporated per RNA molecule. DEAE-cellulose chromatography of product RNA after alkaline hydrolysis suggested that the 5' terminus was the site of methylation.
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PMID:Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme. 105 77

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.
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PMID:Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA. 219 63

U6 small nuclear RNA (snRNA) is a required component in the splicing of eukaryotic pre-mRNAs. Mammalian U6 snRNA was synthesized in vitro by T7 RNA polymerase and purified on polyacrylamide gels. This U6 RNA, with pppG on its 5' end, was accurately capped to CH3-O-pppG, when incubated with HeLa cell extract and this capping was dependent on the capping signal present within the U6 snRNA. When gamma-32P-labeled U6 RNA was used as a substrate, the U6 cap formed in vitro retained this labeled gamma-phosphate, indicating that the cap formation involves the methylation of the gamma-phosphate incorporated during transcription. U6 snRNAs with ppG or pG as their 5' ends, were not capped in this in vitro capping system. Capping of U6 snRNA in vitro requires at least two components, a heat-labile component and S-adenosylmethionine as a methyl group donor. The data presented here show that capping of U6 snRNA can be uncoupled from transcription and that the mechanism of U6 snRNA cap formation differs markedly from the capping mechanism of mRNAs and other U snRNAs where capping is cotranscriptional. While many methyltransferases have been characterized earlier, this is the first report of a methyltransferase that is specific to phosphate residues. This in vitro capping system will be useful for purification and studies on the U6 snRNA sequence-dependent methyltransferase activity.
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PMID:Capping of U6 small nuclear RNA in vitro can be uncoupled from transcription. 234 79


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