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)

The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion G----T, leading to amino acid substitution Val146----Phe. This mutational change marks the second domain of the beta subunit involved in rifampicin binding.
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PMID:Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli. 638 26

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.
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PMID:Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs. 753 29

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.
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PMID:A mutant T7 RNA polymerase as a DNA polymerase. 755 4

Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
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PMID:Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. 764 61

We have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.
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PMID:Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates). 767 87

Reverse transcriptase and polymerase chain reaction methods were used to amplify and clone actin cDNAs from the chlorophylls a + C-containing unicellular alga, Emiliania huxleyi (Prymnesiophyta). Actins in E. huxleyi are defined by a gene family containing at least six distinct coding regions that were derived from relatively recent gene duplications. Five of the coding regions (types 1, 2, and 4-6) varied only among synonymous codons. A nonsynonomous change in a sixth coding region (type 3 actin) produced a serine-to-phenylalanine replacement. The G + C composition of third positions in E. huxleyi actin genes is 98%, which contrasts with the mean value of 50% G + C content for first and second positions. Distance-matrix and parsimony analyses of actin genes identified the prymnesiophytes as a photosynthetic lineage that is not already related to other eukaryotic algal groups.
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PMID:Isolation and molecular phylogenetic analysis of actin-coding regions from Emiliania huxleyi, a Prymnesiophyte alga, by reverse transcriptase and PCR methods. 768 35

The purified TyrR protein and phenylalanine were sufficient to activate in vitro transcription from the tyrP promoter by wild-type RNA polymerase. Such TyrR-mediated activation did not occur when the mutant alpha 235 RNA polymerase was used, indicating that TyrR is a class I transcription activator.
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PMID:The TyrR protein of Escherichia coli is a class I transcription activator. 779 38

The yeast shi mutation affects the spacing between the TATA promoter element and transcription initiation sites; for the H2B and ADH1 genes, a series of start sites located approximately 50-80 bp downstream of TATA is used in addition to the wild-type initiation sites located at around 100 bp from TATA (1). Here, the yeast SHI wild-type gene has been isolated by complementation and shown to be identical to RPB9, the gene encoding a small subunit of RNA polymerase II. A point mutation in the shi gene, changing a cysteine residue in a putative zinc ribbon motif into a phenylalanine residue, was demonstrated to permit the observed usage of upstream initiation sites. Deletion of the non-essential SHI gene also results in usage of upstream initiation sites and causes conditional growth defects.
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PMID:Role of a small RNA pol II subunit in TATA to transcription start site spacing. 780 Apr 82

A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of 15N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.
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PMID:A novel method for selective isotope labeling of bacterially expressed proteins. 788 Dec 74

We report the identification of three new alpha-amanitin resistance mutations in the gene encoding the largest subunit of mouse RNA polymerase II (RPII215). These mutations are clustered in a region of the largest subunit that is important for transcription elongation. This same domain has been identified as the site of alpha-amanitin resistance mutations in both Drosophila and Caenarhabditis elegans. The sequences encompassing this cluster of mutations are highly conserved among RNA polymerase II genes from a number of species, including those that are naturally more resistant to alpha-amanitin suggesting that this region of the largest subunit is critical for a conserved catalytic function. The mutations reported here change leucine 745 to phenylalanine, arginine 749 to proline, or isoleucine 779 to phenylalanine. Together with the previously reported asparagine 792 to aspartate substitution these mutations define a potential alpha-amanitin binding pocket in a region of the mouse subunit that could be involved in translocation of polymerase during elongation.
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PMID:Clustered alpha-amanitin resistance mutations in mouse. 789 49


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