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)

An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel filtration indicated a molecular mass of 28 +/- 5 kDa under nondenaturing conditions, suggesting that the native BTF1Y protein is a monomer. BTF1Y was enzymatically cleaved, several peptides were sequenced, and appropriate oligonucleotide probes were synthesized to clone the BTF1Y gene from a yeast genomic library. The BTF1Y gene contains a 720-base-pair open reading frame encoding a protein of 27,003 Da. The recombinant protein expressed in HeLa cells exhibited the same chromatographic characteristics and in vitro transcriptional activity as BTF1Y prepared from yeast extracts, confirming the identity of the gene. Gene-disruption experiments indicated that the yeast BTF1Y gene is a single-copy essential gene.
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PMID:Cloning of the gene encoding the yeast protein BTF1Y, which can substitute for the human TATA box-binding factor. 269 73

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.
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PMID:Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro. 275 42

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.
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PMID:Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein. 297 May 23

The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 amino acids. In the spacer region between ntrA and orf there are no apparent transcription termination or promoter sequences and therefore orf may be co-transcribed with ntrA. Previous authors have proposed that NtrA could act as an RNA polymerase sigma factor but the NtrA amino acid sequence does not show a high level of homology to any known sigma factor. However analysis of sequences of five sigma factors from E. coli and B. subtilis has identified two conserved sequences at the C-terminal end of all these polypeptides. These sequences resemble those found in known site-specific DNA-binding domains and may be involved in recognition of conserved -35 and -10 promoter sequences. A similar pair of sequences is present at the C-terminus of NtrA and could play a role in recognition of ntr-activatable promoters.
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PMID:The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. 299

The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2 h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [35S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo+ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).
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PMID:Inhibition of gene expression of T7-related phages by prophage P1. 304 52

Mitochondrial RNA polymerase of Saccharomyces cerevisiae consists of two different proteins: a core RNA polymerase of 145 kd and a specificity factor of 43 kd, which contributes the capacity to recognize promoters of the various genes encoded in the mitochondrial genome. We purified both components by SDS-PAGE, followed by renaturation to the active state. The two components were used either singly or in combination to study their interactions with promoter-containing DNA fragments. The core component showed random and weak interaction with DNA, the specificity factor none at all, whereas both components together specifically bound to a promoter. In DNase I footprinting experiments, promoter-bound RNA polymerase protected a short region of DNA flanked by hypersensitivity sites and centred around the position at which RNA synthesis starts. The initial phase of transcription gave rise to specific changes in this footprint: the upstream border remained at the same position up to synthesis of a 4-nt RNA chain, whereas at the downstream border progressive disappearance of hypersensitivity sites took place.
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PMID:Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition. 305 63

The plastoquinone-binding polypeptide D-2 of the reaction center complex of photosystem II in barley has been expressed in vitro using the expression vector pGem3 containing the psbD gene of chloroplast DNA as template. The full length D-2 polypeptide with an apparent mol. weight of 32 kD is a major product when mRNA is transcribed with SP6 RNA polymerase from an insert containing the psbD gene with a 36 bp 5' leader sequence and a 3' tail of 99 bp downstream of the stop codon. Translation of the mRNA had to be done in a rabbit reticulocyte lysate. Translation also occurred from the internal AUG codons giving rise to truncated D-2 polypeptides with sizes of 30, 25 and 17 kD. They are immunoprecipitable with antisera either raised against amino acid residues 235 to 241 or against 345 residues of the D-2 polypeptide. A truncated translation product of 12 kD probably initiated at codon 247 is not recognized by either antiserum. An additional immunoprecipitable protein with an apparent molecular weight of 46 kD is observed by SDS-PAGE and is interpreted as a homomeric aggregate of D-2 polypeptides. Upon addition of methanol solubilized 2,6 dichloro-p-benzoquinone or other quinones a preferential translation of the full-length and truncated D-2 polypeptides is observed. The use of in vitro synthesized D-2 polypeptides for studies of binding quinones, other electron carriers and chlorophyll chromophores is discussed.
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PMID:In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley. 307 64

The partially purified RNA polymerase II from chicken leukemia cells (Chuang R. Y., Chuang L. F. & Israel M. (1986) Biochem. Pharmacol. 35, 1293-1297) contained multiple subunits with molecular masses (in Da) ranging from 220,000 to 24,000, as shown by SDS-polyacrylamide gel electrophoresis. The enzyme was further purified through phosphocellulose column and fractions containing the enzyme activity were collected and concentrated 400-fold through a microconcentrator. The microconcentrator contained a membrane with a molecular weight cutoff around 30,000 and, hence, removed the 24,000 Da polypeptide from the enzyme. It was found that the resulting enzyme retained all the catalytic activity as compared to the enzyme preparation before the concentration step, suggesting that the stoichiometric amount of the 24,000 Da polypeptide is not required for RNA synthesis activity with a denatured DNA template.
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PMID:The 24,000 Da subunit is not required for the RNA synthesis activity of chicken leukemia RNA polymerase II. 314 27

DNA-dependent RNA polymerase has been purified from gram-positive Lactobacillus acidophilus and found to be composed of 4 protein subunits, alpha, beta, beta', and sigma, with molecular weights of 40,000, 150,000, 135,000, and 45,000 kD, respectively, estimated on the basis of SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibits optimal activity in the presence of Mn2+, while Mg2+ shows only a slight effect. The L. acidophilus enzyme transcribes several Escherichia coli promoters examined so far, such as promoters of trp operon, lacUV5, and bla P3 from pBR322, whereas it lacks the ability to recognize bla P1 and tet P2 promoters from pBR322. Thus, the specificity of L. acidophilus RNA polymerase in recognizing the promoters is somehow different from that of the E. coli enzyme. By means of an in vitro transcription assay system for L. acidophilus RNA polymerase, 2 promoters have been identified in the DNA of an L. acidophilus cryptic plasmid (pRNL5). These promoters possess nucleotide sequences in the -10 region similar to the consensus sequence for the E. coli promoters.
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PMID:Characterization and promoter selectivity of Lactobacillus acidophilus RNA polymerase. 315 Jun 81

After poly(rI).poly(rC) induction of FS-4 fibroblasts, both human interferon-beta (IFN-beta) mRNA and an additional induced RNA class (12S RNA) hybridize to a genomic cosmid clone containing the human IFN-beta gene as well as 35 kbp of flanking sequences. However, this coinduced 12S RNA does not originate from regions in the neighborhood of the IFN-beta gene, but hybridizes to the genomic cosmid clone via repetitive Alu-family sequences. While IFN-beta mRNA rapidly decays after reaching a maximum 2-4 h after induction, this 12S RNA is stably maintained in the fibroblast cell for more than 16 h. Contrary to IFN-beta mRNA, the level of the 12S RNA is not further elevated by superinduction conditions (cycloheximide treatment) during poly(rI).poly(rC) induction. However, subsequent to treatment with the weaker viral inducer Newcastle disease virus (NDV) both IFN-beta and the 12S RNA transcripts are induced to a higher level in the presence of cycloheximide. Cell-free translation of hybrid-selected 12S RNA leads to detection of an induced protein of 14 kDa. cDNA cloning reveals that the 12S RNA contains part of an Alu-family sequence in the 5'-untranslated region. The 12S RNA is probably not an RNA polymerase III transcript and codes for a protein of 9 kDa (as monitored by in vitro cell-free translation). This discrepancy in molecular mass can be attributed to a retarded migration of the protein in SDS/PAGE.
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PMID:Alternative mechanisms for gene activation induced by poly(rI).poly(rC) and Newcastle disease virus. 320 96


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