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)

Lys-172 residue of bacteriophage T7 RNA polymerase (T7RP) was substituted for Leu and Gly and Lys-172, Arg-173 were deleted by the site-directed mutagenesis using synthetic oligonucleotides. The specific activity of all mutant enzymes did not differ significantly from that of the wild-type (w.t.) T7RP while for Gly-172 mutant (G172) it was somewhat lower. Leu-172 (L172) and deletion (DEL172-3) mutants were able to direct RNA synthesis on the templates lacking the T7 promoter. DEL172-3 was not able to synthesize extraneous RNA sequences in addition to the expected run-off transcripts. L172 and DEL172-3 mutants revealed altered template specificity toward various DNA templates and showed the lower stability of enzyme-promoter complexes. The possible role of Lys-172 likely belonging to an interdomain "stretch" is discussed.
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PMID:[Site-specific mutagenesis of residue Lys-172 of phage T7 RNA polymerase: characterization of transcription properties of mutant proteins]. 147 Jan 70

The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.
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PMID:Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli. 154 41

We demonstrate that four different proteins from calf thymus are able to restore splicing in the same splicing-deficient extract using several different pre-mRNA substrates. These proteins are members of a conserved family of proteins recognized by a monoclonal antibody that binds to active sites of RNA polymerase II transcription. We purified this family of nuclear phosphoproteins to apparent homogeneity by two salt precipitations. The family, called SR proteins for their serine- and arginine-rich carboxy-terminal domains, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kD. Microsequencing revealed that they are related but not identical. In four of the family members a repeated protein sequence that encompasses an RNA recognition motif was observed. We discuss the potential role of this highly conserved, functionally related set of proteins in pre-mRNA splicing.
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PMID:SR proteins: a conserved family of pre-mRNA splicing factors. 157 77

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal growth factor receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to 15-fold compared with basal levels) 15-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected fibroblast lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal growth factor. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.
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PMID:Interleukin-1 represents a new modality for the activation of extracellular signal-regulated kinases/microtubule-associated protein-2 kinases. 165 5

RNA polymerase, the principal enzyme of gene expression, possesses structural features conserved in evolution. A substitution of an evolutionarily invariant amino acid (Lys1065----Arg) in the beta subunit of Escherichia coli RNA polymerase apparently disrupts its catalytic center. The mutant protein inhibited cell growth when expressed from an inducible promoter. The assembled holoenzyme carrying the mutant subunit formed stable promoter complexes that continuously synthesized promoter-specific dinucleotides but that did not enter the elongation step. The mutant polymerase inhibited transcription by blocking the access of the wild-type enzyme to promoters.
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PMID:Blocking of the initiation-to-elongation transition by a transdominant RNA polymerase mutation. 169 14

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.
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PMID:Mutational analysis of the galactose binding ability of recombinant ricin B chain. 171 62

Monoclonal antibody 104 recognizes a subset of amphibian nuclear granules (B-snurposomes) and active sites of RNA polymerase II transcription in vertebrates and invertebrates. Monoclonal antibody 104 reacts with a set of nuclear serine- and arginine-rich phosphoproteins (SR family) with strikingly conserved apparent molecular masses. The most abundant family members in human (SRp33) and Drosophila (SRp55) cell lines can replace one another as essential splicing factors in a human cell-free system. Each of these polypeptides can functionally replace human SF2, an essential splicing factor that also regulates 5' splice site selection of alternatively spliced pre-mRNAs in vitro. Drosophila SRp55 also functions as an alternative splicing factor in the human cell-free system. Analysis of cloned cDNAs shows that SRp55 and SF2 are highly related and reveals regions of similarity to genetically defined regulators of alternative splicing in Drosophila. These results suggest that the conserved SR family of phosphoproteins, which includes SRp55 and SF2, is involved in constitutive pre-mRNA splicing and in the specificity of alternative splice site selection.
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PMID:Two members of a conserved family of nuclear phosphoproteins are involved in pre-mRNA splicing. 174 84

The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta subunit of the Escherichia coli enzyme (Lys1065 and His1237) were mapped close to the binding site of the priming substrate of the reaction. Surprisingly, the mutational substitution of these residues (Lys1065----Arg and His1237----Ala) did not inactivate the catalytic function, but inhibited transition from the initiation to the elongation stage of transcription.
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PMID:Mapping of the priming substrate contacts in the active center of Escherichia coli RNA polymerase. 174 65

Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.
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PMID:Cassette mutagenesis implicates a helix-turn-helix motif in promoter recognition by the novel RNA polymerase sigma factor sigma 54. 178 87

The nusA11 mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coli. Suppressor mutations that restored growth of nusA11 mutant cells were isolated and named sna mutations. The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the beta' subunit of RNA polymerase. sna-10 complemented the defect in tR1 termination caused by nusA11 and by itself stimulated termination of transcription at the lambda tR1 terminator. sna-10 is specific to the nusA11 allele and unable to suppress cold-sensitive growth of the nusA10 mutant. nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence. During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses. The revised sequence revealed that nusA1 and nusA11 are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively. The intragenic suppressor study indicated that the nusA11 mutation can be suppressed by changing the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine.
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PMID:Genetic interaction between the beta' subunit of RNA polymerase and the arginine-rich domain of Escherichia coli nusA protein. 184 65


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