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)

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.
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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.
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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.
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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.
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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.
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PMID:Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus. 778 76

In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase. These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-[(5-azido-2-nitrobenzoyl)oxy]succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA). We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol. The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase. In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase. With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.
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PMID:Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha. 791 30

The repetitive C-terminal domain (CTD) of RNA polymerase (RNAP) II is extensively phosphorylated concomitant with the initiation of transcription and must be dephosphorylated before RNAP II can begin another round of transcription. A CTD phosphatase was purified more than 7,500-fold from a HeLa cell extract. SDS-polyacrylamide gel electrophoresis shows a predominant protein of 205 kDa and a less abundant protein of 150 kDa co-eluting with the CTD phosphatase activity. Sedimentation and gel filtration analysis suggest that CTD phosphatase has an elongated structure with a M(r) of 200,000. This enzyme is a type 2C phosphatase in that it requires Mg2+ for activity and is resistant to okadaic acid. CTD phosphatase appears to processively dephosphorylate the CTD and is specific in that it does not dephosphorylate phosphorylase a, the alpha or beta subunits of phosphorylase kinase or RNAP II phosphorylated with casein kinase II. CTD phosphatase dephosphorylates RNAP IIO purified from calf thymus or generated in vitro by two previously described CTD kinases. These results suggest that CTD phosphatase has the properties expected for a protein phosphatase that catalyzes the conversion of RNAP IIO to RNAP IIA and may play a key role in the transcription cycle of RNAP II.
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PMID:Purification and characterization of a phosphatase from HeLa cells which dephosphorylates the C-terminal domain of RNA polymerase II. 792 41

Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human colon and ileocecum. Two separate loci (tia and tib) that direct noninvasive E. coli HB101 to adhere to and invade intestinal epithelial cells have previously been cosmid cloned from ETEC H10407. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions from tib-positive HB101 shows that the tib locus directs the synthesis of a 104-kDa outer membrane protein (the TibA protein). The tib locus was subcloned to a maximum of 6.7 kb and mutagenized with transposon Tn5. Production of TibA was directly correlated with the capacity of the subclones and Tn5 mutants to invade and adhere to epithelial cells, suggesting that TibA was required for these phenotypes. The position and direction of transcription of the tibA gene were identified by complementation and in vivo T7 RNA polymerase-promoter induction experiments. The role of the tib locus in epithelial cell invasion was confirmed by the construction of chromosomal deletion derivatives in H10407. These deletion mutants invaded epithelial cells at about 15% of the parental level and were fully complemented by plasmids bearing the tib locus. The size and function of the TibA protein are similar to those of invasin from Yersinia pseudotuberculosis (103 kDa). However, a tib probe did not hybridize with the gene encoding invasin. Hybridization analyses of genomic DNA from a wide variety of pathogenic and nonpathogenic bacteria, including Salmonella, Shigella, Yersinia, and Escherichia species, indicate that the tib locus is unique to specific ETEC strains.
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PMID:Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. 803 17

Region 2 of eubacterial sigma factors is highly conserved and the subdomain 2.4 is involved in -10 promoter recognition. An evolutionary conserved "RpoD box" has been identified at the junction of subdomain 2.3/2.4 in class I and class II sigma factors and there are two tryptophan residues at position 433 and 434 which can be used as intrinsic fluorescent markers to study their structure-function relationship. Site-directed mutagenesis of these two tryptophan residues has been carried out to generate three variants of sigma 70 of Escherichia coli RNA polymerase. These are W433F, W433G and W434G. sigma 70-W433F is found to be indistinguishable from the native sigma factor by both structural and functional analysis. sigma 70-W433G shows anomalous mobility on SDS-PAGE like the native sigma factor, is alpha-helical in conformation (50% helicity) although found to be less active in total transcription when reconstituted with core RNA polymerase. Free sigma 70-W434G, unlike the native sigma factor, shows the expected mobility of a 70 kDa protein on SDS-PAGE and has 20% helicity. Time-resolved fluorescence analysis indicates that free sigma 70-W434G has DNA binding ability, and displays a normal abortive initiation reaction but a decreased level of productive transcription after reconstitution with core RNA polymerase. A model is proposed in which tryptophan at position 434 interacts with the hydrophobic 1.1 domain of sigma 70 giving rise to the stability of the protein under denaturing conditions.
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PMID:A point mutation at the junction of domain 2.3/2.4 of transcription factor sigma 70 abrogates productive transcription and restores its expected mobility on a denaturing gel. 807 73

The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes. We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL). Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family. We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family. Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system. SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E. coli cells. This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc. The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum. These differences may be due to a number of factors, which will be discussed.
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PMID:The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli. 812 94


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