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)

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved.
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PMID:Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins. 272 21

We have used NMR to study the structure of the yeast tRNA(Phe) sequence which was synthesized by using T7 RNA polymerase. Many resonances in the imino 1H- spectrum of the transcript have been assigned, including those of several tertiary interactions. When the Mg2+ concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNA(Phe). The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNA(Phe) [Sampson, J. R., & Uhlenbeck, O. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1033-1037], suggesting that the structure of the two molecules must be similar. In the absence of Mg2+ or at [tRNA]:[Mg2+] ratios less than 0.2, the transcript does not adopt the native structure, as shown by both chemical shifts and NOE patterns. In these low Mg2+ conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript. NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation.
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PMID:Structure of an unmodified tRNA molecule. 277 36

Human transcription factor IIIC (TFIIIC) is an initiation factor required for the in vitro transcription of 5 S RNA, tRNA, and adenovirus viral-associated (VA) RNA genes by RNA polymerase III. A TFIIIC activity which complemented purified TFIIIB and RNA polymerase III fractions for VA transcription was highly purified from cultured HeLa cells. This activity copurified through all chromatographic procedures, including B-block oligodeoxynucleotide affinity chromatography, with the two forms of TFIIIC detected by gel mobility shift assays with the VA gene (Hoeffler, W.K., Kovelman, R., and Roeder, R.G. (1988) Cell 53, 907-920). Both specific binding activity to the VAI gene and TFIIIC transcription activity were inhibited by the alkylating agents diisopropyl fluorophosphate, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), and N-ethylmaleimide, and to a lesser extent by N alpha-p-tosyl-L-lysine chloromethyl ketone, whereas neither activity was inhibited by phenylmethylsulfonyl fluoride. These data suggest further that the DNA binding and transcription assays scored the same protein(s). TPCK and N-ethylmaleimide inactivated TFIIIC solely through thiol group modification, since prior modification with the reversible thiol reagent 2,2'-dithiopyridine prevented permanent inactivation. The involvement of reduced thiol groups in the specific binding of TFIIIC to the VAI gene was further indicated by an increase in TFIIIC binding activity upon addition of dithiothreitol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mr = 126,000 polypeptide both eluted from a B-block oligodeoxynucleotide affinity column with the DNA binding and transcription activities of TFIIIC and was specifically cross-linked by UV to a 5-bromo-2-deoxynucleotide-substituted B-block oligodeoxynucleotide. The near identity of the TFIIIC molecular weight determined by gel filtration on SOTA Phase GF 200 (Mr = 140,000) suggests that TFIIIC in solution (in the presence of 0.3 M NaCl at pH 7.0) consists of a single polypeptide which is fairly globular in nature.
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PMID:Human transcription factor IIIC (TFIIIC). Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding. 280 67

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.
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PMID:Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues. 285 71

Moraxella bovis pili have been shown to play a major role in both infectivity and protective immunity of bovine infectious keratoconjunctivitis. Sonicated M. bovis DNA from the piliated strain EPP63 was inserted into the vector lambda gt11 with EcoRI linkers. Recombinant phage were screened with an oligonucleotide probe based on the amino-terminal portion of the DNA sequence of a Neisseria gonorrhoeae pilin gene. Two candidate phages produced a protein that comigrated with EPP63 beta pilin in sodium dodecyl sulfate-polyacrylamide gels and bound anti-pilus antisera. The 1.9-kilobase insert from one of these, lambda gt11M182, was subcloned in both orientations into pBR322, forming the plasmids pMxB7 and pMxB9, both of which produced beta pilin, as did pMxB12, a HindIII deletion derivative of pMxB7. In HB101(pMxB12), the M. bovis pilin protein was shown to be primarily localized in the inner membrane. The entire 939-base-pair insert of pMxB12 was sequenced, revealing a ribosome binding site just upstream of the coding region and an AT-rich region further upstream containing some potential RNA polymerase recognition sites. The translation of the sequence predicts a six-amino-acid leader sequence preceding the phenylalanine that begins the mature protein. Codon usage analysis of the M. bovis beta pilin gene revealed greater use of the CUA codon for leucine than usual for a well-expressed Escherichia coli gene. Comparisons of the M. bovis EPP63 beta pilin protein sequence with other pilin gene sequences are presented.
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PMID:Cloning and sequencing of a Moraxella bovis pilin gene. 286 Nov 94

Ts-phenotype of the E. coli rho-factor mutant rho 15 is suppressed by two rifampicin-resistance mutations, rhoB1019 resulting in a single amino acid substitution Val146----Phe and rhoB268 resulting in a single substitution Gln513----Leu in beta-subunit of the E. coli RNA polymerase. Rifampicin-resistance mutations rhoB255 (Asp516----Val), rhoB1016 (Asp516----Asn), rhoB1001 (His526----Tyr), rhoB1004 (Ser531----Phe), rhoB1005 (Pro564----Leu), and streptolydigin-resistance' mutation rhoB1018 (double substitution Gly544----Asp and Phe545----Ser) do not suppress the rho15 mutation.
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PMID:[Amino acid substitutions in the beta-subunit of RNA-polymerase from E. coli compensating for mutation-induced damage of the rho termination factors]. 305 19

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.
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PMID:A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties. 319 21

A bacteriophage gamma Ch4A clone containing a 22-kb rat DNA insert was isolated and found to contain a solitary tRNA(Phe)GAA gene and, 436 bp downstream of it, an Alu-like element. The nucleotide sequence of a 1141-bp DNA fragment containing these genes was determined. The rat tRNA(Phe)GAA gene, with the exception of an additional A in the extra arm, has a sequence identical to that of a rabbit liver tRNA(Phe). The Alu-like element belongs to the rodent B2 family of short interspersed repetitive nucleotide sequences. This repetitive element, B2Phe, is flanked by 12-bp direct repeats, contains an internal split promoter (block A and block B) for RNA polymerase III and is devoid of an A-rich segment at the 3' end. Like other members of the B2 family, the B2Phe element presents 64% sequence homology with rat serine tRNA and contains a serine (GCT) anticodon. Both tRNA(Phe)GAA gene and B2Phe element were found to be transcriptionally active in HeLa cell and Xenopus oocyte nuclear extracts. The tRNA(Phe) gene transcripts were processed during the course of transcription to form mature-size tRNA(Phe). The transcription efficiency of the B2Phe element was found to be an order of magnitude higher than that of the tRNA(Phe) gene. Competition experiments demonstrate that the B2Phe DNA can form a more stable transcription complex than the tRNA(Phe) gene and compete with it for binding of transcription factors.
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PMID:Nucleotide sequence and transcription of a rat tRNA(Phe) gene and a neighboring Alu-like element. 323 68

Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.
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PMID:Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. 350 89

A 13.8-kb fragment of human DNA isolated from a human lambda Charon-4A DNA library was found to contain four human tRNA genes. Nucleotide sequence analysis of approx. 3.7 kb of this segment of human DNA identified two lysine tRNA(UUU) genes identical in coding sequence to a previously reported human lysine tRNA gene [Roy et al., Nucl. Acids Res. 10 (1982) 7313-7322]. The other two tRNA genes were phenylalanine tRNA(GAA) genes, the first to be isolated from a mammalian source. These phenylalanine tRNA(GAA) genes were identical in sequence with the exception of a G/A polymorphism at coordinate 57. None of these tRNA genes contains introns. The tRNA(UUULys) and tRNA(GAAPhe) genes are organized in alternating order and are irregularly spaced, by intergenic regions of approx. 1.0, 2.6 and 5.0 kb, and randomly oriented. There was no evidence to indicate that any of these genes arose by gene duplication, since flanking sequence homology was limited to the putative RNA polymerase III termination signals in the 3'-flanking regions. A mature tRNA-sized product was identified following the transcription of each tRNA gene in a homologous in vitro transcription system. Interestingly, different levels of transcriptional activity of the three identical lysine tRNA genes were observed, suggesting modulation of tDNA expression by extragenic sequences. In addition, a minimum of eight regions of homology to Alu-type repetitive elements were detected in this human DNA fragment, one of which was located 53 bp upstream from a tRNA(GAAPhe) gene.
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PMID:Analysis of a human gene cluster coding for tRNA(GAAPhe) and tRNA(UUULys). 367 37


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