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 developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.
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PMID:A simple assay for determining the capping efficiencies of RNA polymerases used for in vitro transcription. 226 28

The complete nucleotide sequence of white clover mosaic virus (WCIMV) strain O has been determined and compared to the WCIMV strain M sequence. The two virus strains show 12% divergence at the nucleotide level and 3% divergence at the amino acid level (open reading frames 1 to 5). Two open reading frames, 10 and 21 kDa, identified in the strain M sequence were not found in the strain O sequence. High levels of infectious RNA transcripts were produced from a full-length cDNA clone of strain O constructed downstream of the SP6 RNA polymerase promoter (greater than 2 micrograms RNA/micrograms template DNA). Symptoms characteristic of WCIMV developed when host plants were inoculated with SP6 RNA transcripts. The specific infectivity of transcript RNA produced in the presence of m7GpppG was similar to that of virion RNA. The SP6 transcripts lacked 5' nonviral nucleotides, but contained 6, 14, or 198 3' nonviral nucleotides (derived from the pUC19 vector). Transcripts were infectious without m7GpppG, or with long nonviral 3' nucleotide stretches. Progeny virus RNA from infections initiated with transcripts containing 198 nonviral 3' nucleotides did not maintain these sequences.
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PMID:Infectious transcripts and nucleotide sequence of cloned cDNA of the potexvirus white clover mosaic virus. 235 51

We determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100,383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.
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PMID:The gene for Klebsiella bacteriophage K11 RNA polymerase: sequence and comparison with the homologous genes of phages T7, T3, and SP6. 237 Aug 50

Mammals contain a family of five closely related H1 histone variants (H1a-e) as well as two less closely related forms, H10 and H1t. We have sequenced a rat genomic clone that encodes one of the standard H1 variants. An RNA transcript of the gene was made with bacteriophage SP6 RNA polymerase and translated in a cell-free system. The protein synthesized in vitro was identified as variant H1d by its electrophoretic mobility.
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PMID:Isolation of a genomic clone encoding the rat histone variant, H1d. 237 70

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.
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PMID:Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA. 241 34

Four RNA fragments of approximately 1,000 to 1,200 nucleotides, representing both the 5' and 3' termini of poliovirus plus- and minus-strand RNAs, were generated by transcription of poliovirus cDNA by using bacteriophage SP6 RNA polymerase. The copying of these templates by the poliovirus replicase invariably produced RNA products approximately twice the size of the templates. In experiments with templates uniformly labeled with 32P it was shown that some of the apparently double-length products were generated by extension from an internal site of the template. Filter hybridization of the labeled in vitro-synthesized products with various unlabeled templates suggested a second mechanism by which double-length molecules could be synthesized; the results can be best explained by de novo synthesis of the first strand by copying of the template RNA, followed by snap-back of the newly synthesized RNA, generating a template-primer structure for the synthesis of the second strand. Highly purified poliovirus replicase was able to support the synthesis of double-length RNA products in response to these templates. These reactions did not require host factor. In contrast, synthesis of genome-length copies of poliovirion RNA by the same replicase was absolutely dependent on added host factor. The synthesis of double-length RNA products did not require either the 3'-terminal poly(A) of plus RNA or sequences within the 3' termini of both plus- and minus-strand RNAs.
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PMID:Mechanism of in vitro synthesis of covalently linked dimeric RNA molecules by the poliovirus replicase. 242 94

The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5' splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5' and 3' splice sites independently show that splicing occurs in two separable steps. The 3' splice site was found to be susceptible to site-specific hydrolysis leaving a hydroxyl terminus. This is interpreted as an indication that the 3' splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5' splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); transesterification by oligopyrimidines or by the 5' exon (which terminates with C-U-C-U-C-UOH) at the site following the 3'-terminal guanosine residue of the intervening sequence; and specific hydrolysis at the splice sites. One of the products of the reactions at the 3' splice site is a molecule that contains the 5' exon still attached to the intervening sequence. It has a 3'-terminal GOH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5' splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3'-terminal G of the intervening sequence.
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PMID:New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing. 243 Nov 51

Two plant introns along with flanking exon sequences have been isolated from an amylase gene of wheat and a legumin gene of pea and cloned behind the phage SP6 promoter. Pre-mRNAs produced by in vitro transcription with SP6 RNA polymerase were tested for their ability to be spliced in a HeLa cell nuclear extract. The plant introns were accurately spliced and the predicted splice junctions were used. Lariat RNAs were observed as both intermediates and final products during the splicing reaction. The branch points were mapped to adenosine residues lying within sequences that showed good homology to the animal branch point consensus. Consensus sequences for the 5' and 3' splice junctions and for putative branch point sequences of plants were derived from an analysis of 168 plant intron sequences.
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PMID:Accurate in vitro splicing of two pre-mRNA plant introns in a HeLa cell nuclear extract. 243 97

Satellite 2 of the newt, Notophthalmus viridescens, is a 330 bp tandemly repeated sequence scattered throughout the genome. Cytoplasmic transcripts homologous to satellite 2 are found in a variety of tissues. Most of the transcripts correspond precisely in length to the DNA repeat unit or to whole multiples of that repeat. We show here that dimer-sized satellite 2 transcripts, synthesized with SP6 RNA polymerase from a plasmid clone, undergo site-specific, self-catalyzed cleavage in vitro. The reaction proceeds at neutral pH and requires Mg++ but no other cofactor or energy source. The cleavage products have 5'-hydroxyl and 3'-phosphate groups, at least some of which are in the form of 2',3'-cyclic phosphates. In this respect the reaction resembles the self-cleavage of certain small, infectious RNAs found in plants. Furthermore, the in vitro cleavage of satellite 2 transcripts occurs within a sequence that is homologous to the conserved cleavage site of the infectious RNAs. The existence of monomer and multimer transcripts in the cell suggests that the monomer may arise by site-specific cleavage of long primary transcripts. However, the 5' end of the cellular monomer is 46 or 47 bases upstream of the in vitro cleavage site, suggesting that factors in the cell may modify the cleavage reaction.
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PMID:Self-cleaving transcripts of satellite DNA from the newt. 243 49

Since previous studies have suggested that the mammalian protamine mRNAs are translated poorly in cell-free systems, we directly measured the efficiency of translation of mouse protamine 1 mRNA. We found that mouse testis poly(A)+ mRNA stimulates the synthesis in the wheat germ and reticulocyte cell-free systems of three prominant translation products which can be resolved by electrophoresis through acid urea polyacrylamide gels containing 8 M urea. These translation products have been identified as testis-specific protein, protamine 1, and the precursor to protamine 2 by several criteria, including labeling with amino acids, [35S]cysteine, and [3H]leucine, which are known to be specific to some of these proteins from the nucleotide sequences of recombinant DNAs. Surprisingly, the mobility of the testis-specific protein translation product is slightly reduced and the mobility of both protamine translation products is drastically reduced unless the extracts of cell-free translations are coelectrophoresed with the appropriate carrier. The fraction of [35S]cysteine- labeled protamine 1 translation product was compared with the fraction of testis poly(A)+ mRNA as protamine 1 mRNA which we measured in dot blots with the use of an SP6 RNA polymerase transcript for protamine 1. The results demonstrate that protamine 1 mRNA is translated only slightly less efficiently than the average testis poly(A)+ mRNA.
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PMID:Translation of mouse testis poly(A)+ mRNAs for testis-specific protein, protamine 1, and the precursor for protamine 2. 244 49


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