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)

Reovirus mRNAs synthesized by the virion-associated RNA polymerase contain a 5'-terminal cap that is added to nascent transcripts by polypeptide lambda 2, a structural component of virions encoded by double-stranded RNA genome segment L2. The complete, 3916-nucleotide sequence of a full-length reovirus type 3 L2 DNA clone was determined by the dideoxy chain terminator method. The sequence has a single long open reading frame extending from the second A-T-G at nucleotide 14 to a termination codon at position 3881. On this basis, the 1289-amino acid sequence of polypeptide lambda 2, the reovirus mRNA guanylyltransferase, was deduced and compared to other GTP-binding proteins. Two different, lysine-containing lambda 2 peptide sequences closely resemble predicted amino acid stretches in vaccinia virus guanylyltransferase and potentially form part of active sites in the viral mRNA capping enzymes.
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PMID:Complete nucleotide sequence of reovirus L2 gene and deduced amino acid sequence of viral mRNA guanylyltransferase. 282 87

We have shown that an extract made from HeLa cells harvested 6 hr after infection with vaccinia virus can transcribe a duplex DNA template containing a late viral gene. S1 nuclease analyses using genomic and synthetic probes indicated that the 5' ends of RNA synthesized in vitro are similar to those of RNA made in vivo and contain 5' poly(A) sequences contiguous with the translation initiation codon. Kinetic analysis of RNA synthesized in vitro demonstrated that a correctly initiated and 5' polyadenylylated product appeared within 5 min after transcription reactions were started. A cis-splicing mechanism of poly(A) addition can be ruled out because the DNA template used in vitro had no poly(dT) sequence and could contain as few as 37 base pairs upstream of the start of the RNA. In addition, we found that a point mutation in the first of two consecutively encoded adenylate residues preceding the ATG initiation codon abolished transcription in vitro. These data are consistent with at least three models: (i) RNA polymerase initiates RNA synthesis with a run of adenylate residues; (ii) a poly(A) primer is used for initiation; or (iii) the poly(A) leader is rapidly and efficiently attached to the RNA by ligation.
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PMID:In vitro synthesis of vaccinia virus late mRNA containing a 5' poly(A) leader sequence. 282 58

Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.
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PMID:Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors. 282 73

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.
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PMID:Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. 283 68

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.
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PMID:Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. 284 43

The nucleotide sequence of segment 1 of the double stranded RNA genome of bluetongue virus serotype 10 (BTV-10), encoding the largest viral core protein, VP1, has been determined. Linear sequence analysis of the predicted amino acid sequence of the 149-K Da protein, a putative component of the viral RNA-directed RNA polymerase, revealed extensive homology with the vaccinia virus 147K Da DNA-directed RNA polymerase subunit. Similar homologies were detected between the VP1 polypeptide and the beta chain subunit of Escherichia coli and common tobacco chloroplast RNA polymerases, yeast RNA polymerase II and III and fruit fly polymerase II.
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PMID:Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species. 285 May 42

We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively. Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations. Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus. The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus.
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PMID:Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. 291 Nov 20

We have carried out detailed phenotypic characterization of five temperature-sensitive (ts) mutants of vaccinia virus, the ts lesions of which have previously been mapped to two different subunits of the viral RNA polymerase. We have also attempted to determine the mechanism of temperature sensitivity in these mutants. Phenotypic characterization of each of the mutants showed that at the nonpermissive temperature, all five mutants exhibited normal levels of early viral mRNA and protein synthesis, but for an extended period of time, all mutants accumulated normal levels of DNA in abnormally large pools in the cell cytoplasm; all mutants were defective in the synthesis of late viral mRNA and proteins and in viral morphogenesis. In an attempt to address the mechanism of temperature sensitivity in these mutants, we measured the effect of a temperature shift on the ability of the mutants to direct late viral protein synthesis. If infected cells were shifted down from a nonpermissive temperature late during infection, late protein synthesis was initiated after a lag period of 1 to 2 h. If infected cells were shifted up from a permissive temperature early during infection, late protein synthesis continued to be defective. If infected cells were shifted up to the nonpermissive temperature after late protein synthesis had commenced, late protein synthesis was maintained at the nonpermissive temperature at the level observed when the temperature was shifted up. We interpret these results to mean that once a functional RNA polymerase has been assembled at the permissive temperature during a mutant infection, it remains functional at the nonpermissive temperature, but that the ts mutants are defective in the assembly of a newly synthesized RNA polymerase at the nonpermissive temperature. This interpretation implies that the virion RNA polymerase is responsible for early viral transcription and that a newly synthesized RNA polymerase transcribes late viral genes.
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PMID:Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. 291 Nov 21

We have characterized the poxvirus gene encoding the second-largest subunit of the viral DNA-dependent RNA polymerase. This gene, designated rpo132, is located in the HindIII A fragment of the DNA of the Brighton Red strain of cowpox virus. A similar gene is located in the corresponding position in the HindIII A fragment of the DNA of the Western Reserve strain of vaccinia virus. The rpo132 gene is transcribed throughout the viral multiplication cycle. It has two transcriptional start sites; one is operative at late times only, and the other (80 base pairs downstream) is operative both at early times and at late times. Neither early nor late transcripts originating from the latter RNA start site contain long 5'-terminal poly(A) sequences. The rpo132 gene has the capacity to encode primary gene products of two types. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 133-kilodalton (kDa) protein. The RNA transcripts whose 5' ends correspond to the early RNA start site can encode a 132-kDa protein. Transcripts of the latter type are more abundant, suggesting that the 132-kDa protein is the major primary product of this gene. The predicted amino acid sequences of both gene products share extensive similarities with the amino acid sequences of the second-largest subunits of the following enzymes: the RNA polymerase of Escherichia coli, the RNA polymerase II of Saccharomyces cerevisiae, and the RNA polymerase II of Drosophila melanogaster. This result provides further evidence of relatedness between multisubunit DNA-dependent RNA polymerases.
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PMID:The second-largest subunit of the poxvirus RNA polymerase is similar to the corresponding subunits of procaryotic and eucaryotic RNA polymerases. 291 77

A soluble extract from purified vaccinia virus particles has been developed which displays site-specific initiation of transcription on exogenous DNA templates that carry cloned vaccinia virus early gene sequences. Bacterial plasmid vectors with segments of a strongly expressed early region of the vaccinia virus genome were active templates, whether in supercoiled or linear, truncated forms. Correct initiation, corresponding to that found in vivo, was observed for all early genes tested. The involvement of other factors besides the viral RNA polymerase was demonstrated by the loss of specific initiation upon partial purification of the enzyme. Initiation activity was restored by reconstitution of the system with factors lacking polymerase activity. The soluble system retained properties of transcription characteristic of intact viral cores, including (i) similar relative rates of initiation of various genes, (ii) multiple requirement for ATP, (iii) methylation and polyadenylation of transcripts, and (iv) inhibition by a topoisomerase antagonist.
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PMID:A soluble transcription system derived from purified vaccinia virions. 298 38


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