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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 degrees C was the highest temperature at which V. harveyi was able to grow steadily. A shift from 30 degrees C to 39 degrees C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15kDa protein. We showed that a protein of approximately 43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (sigma 32) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.
...
PMID:Characterization of heat-shock response of the marine bacterium Vibrio harveyi. 747 74

The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
...
PMID:Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein. 750 37

The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to three-hundred-fold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M(r) 68,000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M(r) of the enzyme was estimated to be approximately 265,000 by native gel electrophoresis and approximately 305,000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent Km for pyrroline-5-carboxylate was 109 +/- 7.3 microM, whilst that for NAD+ was 43.3 +/- 2.5 microM. Product inhibition by NADH (apparent Ki 0.6mM) was observed. The observed Vmax was 22.0 +/- 1 mol min-1 (mg protein)-1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.
...
PMID:Interaction between primary and secondary metabolism in Streptomyces coelicolor A3(2): role of pyrroline-5-carboxylate dehydrogenase. 755 Oct 40

The C-terminal domain (CTD) of RNA polymerase II (RNAP II) is essential for the assembly of RNAP II into preinitiation complexes on some promoters such as the dihydrofolate reductase (DHFR) promoter. In addition, during the transition from a preinitiation complex to a stable elongation complex, the CTD becomes heavily phosphorylated. In this report, interactions involving the CTD have been examined by protein-protein cross-linking. As a prelude to the study of CTD interactions, the effect of recombinant CTD on in vitro transcription was examined. The presence of recombinant CTD inhibits in vitro transcription from both the DHFR and adenovirus 2 major late promoters, suggesting that the CTD is involved in essential interactions with a general transcription factor(s). Factors in the transcription extract that interact with the CTD were identified by protein-protein cross-linking. Recombinant CTD was phosphorylated at its casein kinase II site, at the C terminus of the CTD, in the presence of [35S]adenosine 5'-O-(thiotriphosphate) and alkylated with azidophenacyl bromide. Incubation of azido-modified 35S-labeled CTD with a HeLa transcription extract followed by ultraviolet irradiation results in the covalent cross-linking of the CTD to proteins in contact with the CTD at the time of irradiation. Subsequent incubation with phenylmercuric acetate results in the transfer of 35S from the CTD to the protein to which it was cross-linked. The two major photolabeled bands have a M(r) of 34,000 and 74,000. The specificity of CTD interactions was demonstrated by a reduction in photolabeling in the presence of unmodified CTD or RNAP II containing an intact CTD (RNAP IIA) but not in the presence of a CTD-less RNAP II (RNAP IIB). The 35S-labeled 34- and 74-kDa proteins comigrate on SDS-polyacrylamide gel electrophoresis with the beta subunit of transcription factor IIE and the 74-kDa subunit of transcription factor IIF, respectively. Moreover, some of the minor 35S-labeled bands comigrate with other subunits of the general transcription factors.
...
PMID:The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa cell transcription extract that comigrate with transcription factors IIE and IIF. 755 97

TCDD is known to induce thymic atrophy in several mammalian species through activation of programmed cell death, or apoptosis. To investigate the time course of events which precede TCDD-induced thymic apoptosis in vitro, experiments were performed with thymocytes isolated from immature rats. Peak accumulation of both total and specifically bound [3H]TCDD was observed at 60 min post incubation. Incubation of cells with 10 nM TCDD resulted in significant increases in RNA polymerase activity and incorporation of [3H]uridine at 30 min, indicating increased RNA synthesis in response to TCDD. TCDD-induced stimulation of [3H]uridine incorporation was not significantly altered in the presence of cycloheximide, while this effect was abrogated in the presence of actinomycin D. Incubation of thymocytes with 10 nM TCDD also stimulated the activity of poly(A)polymerase, the enzyme catalyzing mRNA polyadenylation, at time points beyond 30 min. No significant increases in [35S] incorporation were observed in cells treated with 10 nM TCDD, although analysis of detergent and high salt extracted nuclear proteins by SDS-PAGE and coomassie blue staining revealed the increased abundance of at least two proteins with molecular masses of 52,000 and 42,000 Da, respectively. These studies reveal that thymocyte nuclei rapidly accumulated TCDD in vitro, leading to increased RNA synthesis, poly(A)polymerase activity and protein synthesis. These events correlate closely with the process of programmed cell death.
...
PMID:Early effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat thymocytes in vitro. 768 Jan 43

DNA dependent RNA polymerase from exponentially growing Staphylococcus aureus cells was purified. An SDS-polyacrylamide gel analysis of the most purified preparation revealed that it consists of beta, beta', alpha, and sigma with apparent molecular masses of 151, 147, 42, and 55 kDa, respectively. The sigma subunit cross reacted with a polyclonal antibody against Bacillus subtilis sigma 43. The cross reacting peptide co-migrated with the B. subtilis sigma 43 subunit. The implications of these results are discussed. Promoter specific in vitro run-off transcripts were obtained using the purified enzyme preparation. Specific conditions for the polymerization reaction are defined.
...
PMID:Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus. 769 14

We have previously characterized an RNA polymerase (pol) I transcription factor, E1BF, from rat cells. This protein is immunologically related to Ku autoantigen and is required in pol-I directed transcription of rodent ribosomal RNA gene (rDNA). Glycerol density gradient fractionation and in situ UV cross-linking analysis of the purified factor showed directly that it consists of a heterodimer of 85 and 72 kDa polypeptides. E1BF also interacted with the human core promoter and augmented transcription of human rDNA as much as fivefold in HeLa nuclear extract, whereas transcription from adenovirus major late promoter, CMV or SV40 early promoters by pol II and of U6 and 5S RNA genes by pol III were either unaffected or minimally inhibited by the antibodies. Purified rat E1BF partially restored the suppression of human rDNA transcription by anti-Ku antibodies. Immunoprecipitation of rat cell extract with the anti-Ku antibodies followed by SDS-PAGE of the precipitated proteins and Southwestern analysis showed that E1BF interacts with CPBF, a core promoter binding factor. When the majority of CPBF and E1BF was removed from the reaction mixture by preincubation with a core promoter oligo nucleotide fragment, rDNA transcription was severely impaired. Addition of exogenous CPBF or E1BF to such a reaction resulted in significant restoration of the transcription, whereas inclusion of both factors caused further enhancement of rDNA transcription. These data demonstrate that E1BF is a basal pol I transcription factor that interacts with a core promoter binding factor both physically and functionally, and that is not a general pol II or pol III transcription factor.
...
PMID:E1BF/Ku interacts physically and functionally with the core promoter binding factor CPBF and promotes the basal transcription of rat and human ribosomal RNA genes. 773 47

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
...
PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. Km for E. coli tRNA(2Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 microM. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.
...
PMID:Substrate specificity of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB27. 776 37

A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.
...
PMID:Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus. 778 76


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---