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 class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 2.7.7.6 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.
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PMID:HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes. 125 52

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.
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PMID:Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli. 130 92

We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit.
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PMID:The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection. 131 73

Mammalian RNA polymerase II contains at the C terminus of its largest subunit an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The phosphorylation of this domain is thought to play an important role in the transition of RNA polymerase II from a preinitiation complex to an elongating complex. The unphosphorylated form of RNA polymerase II is designated IIA, whereas the phosphorylated form is designated IIO. In an effort to determine the consequence of C-terminal domain phosphorylation on complex formation, 32P-labeled RNA polymerases IIA and IIO were prepared and examined for their ability to form a stable preinitiation complex on the adenovirus-2 major late promoter in the presence of a reconstituted HeLa cell transcription extract. Preinitiation complexes were formed in the absence of ATP and purified from free RNA polymerase II by chromatography on Sepharose CL-4B. The state of phosphorylation of the largest subunit was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the transcriptional activity was determined by assaying specific transcript formation upon the addition of nucleotides and a competing DNA template. RNA polymerase IIA was recovered in transcriptionally active complexes in reactions in which the input enzyme was RNA polymerase IIA. In reactions with RNA polymerase IIO as the input enzyme, no IIO was recovered in excluded fractions that normally contain preinitiation complex. In reactions with equimolar amounts of RNA polymerases IIO and IIA, purified preinitiation complexes contained almost exclusively RNA polymerase HA. These results support the idea that RNA polymerase II containing an unphosphorylated C-terminal domain preferentially associates with the adenovirus-2 major late promoter. The state of phosphorylation of the C-terminal domain can, therefore, directly influence preinitiation complex formation. We also report here the presence of an activity in HeLa cell extracts that catalyzes dephosphorylation of the C-terminal domain, thereby converting RNA polymerase IIO to IIA. This C-terminal domain phosphatase is specific in that it does not catalyze the dephosphorylation of a serine residue phosphorylated by casein kinase II. The presence of a C-terminal domain phosphatase in in vitro transcription reactions containing RNA polymerase IIO results in the formation of RNA polymerase IIA. This RNA polymerase IIA associates preferentially with preinitiation complexes.
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PMID:The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. 131 3

Mutations were introduced into a cDNA clone of poliovirus resulting in single-amino-acid substitutions within the region of the proposed FG loop of proteinase 3C. RNAs were made by in vitro transcription with T7 RNA polymerase and used to transfect HeLa cells. Virus viability was assessed as indicated by cell lysis. In parallel, RNAs were translated in vitro by using a HeLa cell lysate, and the patterns of the processed poly-proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Replacement of Lys-78, Arg-79, and Glu-81 had apparently no effect on virus viability and on proteolytic processing. In contrast, virus viability was abolished by mutation of Phe-83, Arg-84, Asp-85, Ile-86, and Arg-87. With respect to substitution of Phe-83, Asp-85, and Arg-87, these effects correlated with impaired processing of the 3CD cleavage site, separating 3C and 3D, and, to a lesser extent, of the P1 precursor. Replacement of Arg-84 and Ile-86, on the other hand, did not alter the processing pattern. Thus, the lethal effects in these mutant genomes may not have been caused by impaired processing. A special case was the mutant of Lys-82-Gln. Virus recovered from cells transfected with RNA carrying this mutation always contained an A-to-G transition which resulted in the replacement of glutamine for arginine. Our data suggest that residues in the proposed FG loop of proteinase 3C influence 3CD cleavage and that they are determinants of a function unrelated to proteolytic processing.
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PMID:Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity. 132 54

DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl2. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.
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PMID:Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida. 136 75

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.
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PMID:The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants. 140 Feb 38

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.
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PMID:Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB. 140 60

T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.
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PMID:Transcription dependence of DNA packaging of bacteriophages T3 and T7. 141 5

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.
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PMID:Cyclopropane fatty acid synthase of Escherichia coli: deduced amino acid sequence, purification, and studies of the enzyme active site. 144 40


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