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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.
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PMID:Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. 165 2

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5' terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3' terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.
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PMID:Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene. 179 54

The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetase. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity.
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PMID:RNA minihelices and the decoding of genetic information. 202 14

The two variants of influenza A/Victoria/35/72 (H3N2) virus resistant simultaneously to remantadine, deitiforin, adapromine and amantadine were obtained while passaging the virus in presence of remantadine or deitiforin. Both variants differed from the parental strain in optimal pH for hemolysis, transcriptase activity and in amino acid sequence of M2 protein. Maximal hemolytic activity of the parental strain is registered at pH 5.2, for the variants cultured in the presence of remantadine or deitiforin at pH 5.5 and 5.8, respectively. In contrast to NH4OH, remantadine and deitiforin do not exert inhibition of virus-induced hemolysis. Transcriptase activity of resistant variants is about 50% higher as compared with parental strain (enzyme source--whole virus particles or RNP). The M2 protein of the remantadine variant has 2 amino acid substitutions: 31 (Ser----Asn) and 59 (Met----Leu); the deitiforin variant has 3 substitutions: 14 (Met----Leu), 30 (Ala----Val) and 59 (Met----Leu). The phenotypic resistance of the virus seems to be determined by the mutations in the hydrophobic protein region (30,31); the other substitutions (14,59) may modify conformational structure and functional activity of the viral proteins.
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PMID:[The change in functional activity and primary structure of the M2 protein in variants of the influenza virus resistant to remantadine and deitiforin: common and individual differences from the original strain]. 281

1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0.45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K(m) (ATP) was 0.65mm and K(m) (l-amino acids) was 70mum. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.
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PMID:The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf. 422 1

1. The alanyl-s-RNA synthetase of tomato roots has been purified by ammonium sulphate precipitation, adsorption on calcium phosphate gel and DEAE-cellulose chromatography and its properties have been investigated. 2. Enzyme activity was measured by using the hydroxamate assay, the [(32)P]pyrophosphate-ATP-exchange assay and the [(14)C]alanyl-s-RNA assay. The purified enzyme was specific for l-alanine and was activated by Mg(2+) ions and to a smaller extent by Co(2+) and Mn(2+) ions. It was free from adenosine triphosphatase, pyrophosphatase and ribonuclease, and possessed a specific activity comparable with that of the most highly purified aminoacyl-s-RNA synthetases from animal and microbial systems. 3. The properties of the purified enzyme were similar in many respects to most other highly purified aminoacyl-s-RNA synthetases. It differed, however, in that the pH optimum of the hydroxamate assay was almost the same as that of the pyrophosphate-ATP-exchange assay and in requiring a high concentration of l-alanine for maximum activity (100mumoles/ml.). 4. The purified enzyme was not absolutely specific for tomato-root s-RNA; slight activity was also observed with yeast s-RNA. 5. The properties of this enzyme are fully consistent with the suggestion that the enzymic formation of alanyl-s-RNA proceeds via the intermediate formation of alanyl acyl-adenylate with the elimination of pyrophosphate from ATP. It remains to be shown the extent to which alanyl-s-RNA participates further in subsequent stages of protein synthesis in plants.
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PMID:The purification and properties of the alanyl-transfer ribonucleic acid synthetase of tomato roots. 428 91

The phosphoprotein P of human respiratory syncytial virus (RSV) was expressed in eukaryotic cells in phosphorylated form. Site-directed mutagenesis of the recombinant protein established Ser232 as the major site of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the phosphorylation of Ser237, whereas mainly Ser232 was phosphorylated by a crude cell extract. The P kinase activity in the cell extract exhibited properties characteristic of CKII. While the Ser232,237 to Ala double mutant was nearly completely defective for phosphorylation and transcription, phosphorylation at Ser232, through the use of appropriate P mutant or kinase, activated P protein. Phosphorylation of Ser237 restored activity only to the extent it facilitated phosphorylation of Ser232. Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no effect on the intracellular turnover of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P protein kinase activity. Thus, CKII-mediated phosphorylation of Ser232 appears to be the primary regulator of P protein activity while phosphorylation of Ser237 may be involved in a modulatory role under certain conditions.
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PMID:Phosphorylation of Ser232 directly regulates the transcriptional activity of the P protein of human respiratory syncytial virus: phosphorylation of Ser237 may play an accessory role. 749 65

Previous mutations associated with lecithin:cholesterol acyltransferase (LCAT) deficiency have been identified using genomic DNA. To facilitate mutation analysis, we used cDNA from cultured fibroblasts which were shown to express LCAT mRNA. Using reverse-transcriptase PCR, LCAT cDNA was obtained from a 13-year-old boy with complete LCAT deficiency, characterized by low HDL-C (3 mg/dl), nondetectable initial cholesterol esterification rate, LCAT activity, and minimal LCAT mass (0.16 vs. 5-7.5 micrograms/ml). Sequencing of LCAT cDNA clones identified two mutations. A novel frameshift mutations caused by deletion of cytosine at the third nucleotide position of amino acid 168 (exon 5) predicts a disrupted protein catalytic site by converting Ser181-->Ala and creates a Pvu-II restriction site prior to premature truncation at amino acid 238. A C-->T transition results in a substitution of methionine for threonine at amino acid position 321 and creates an Nla-III restriction site on the maternal allele. Expression studies of mutant LCAT cDNA confirmed the virtual absence of LCAT activity in transfected COS-1 cells. The molecular defect in a young male with complete LCAT deficiency has been identified using fibroblast cDNA.
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PMID:Lecithin: cholesterol acyltransferase deficiency: identification of two defective alleles in fibroblast cDNA. 765 65

A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted HB9 protein has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues.
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PMID:A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues. 791 94

cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned in E. coli. One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing. Then 1479 nucleotides of the 3'-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract. The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.
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PMID:Nucleotide sequence of the coat protein coding region of tulip breaking virus. 807 37


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