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)

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.
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PMID:Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme. 204 17

The human gastric (H+ + K+)-ATPase gene (15 kilobases) was cloned, and its nucleotide sequence was determined. The gene has 22 exons and codes a protein of 1,035 residues including the initiator methionine (Mr = 114,047). A conserved lysine-rich sequence with inserted glycine residues was found near the amino terminus of the enzyme. The phosphorylation site and pyridoxal 5'-phosphate- and fluorescein isothiocyanate-binding residues found in the rat and pig enzymes are also conserved in the human enzyme. The positions of introns in the human (H+ + K+)-ATPase gene are essentially the same as those in the human (Na+ + K+)-ATPase alpha and alpha III subunits; but the first introns of the two enzymes are difficult to align, and unlike in the (Na+ + K+)-ATPase gene, the sixth exon in the (H+ + K+)-ATPase gene is not separated by an intron. Furthermore, the ninth intron is located two bases upstream of the position for the corresponding intron of the (Na+ + K+)-ATPase alpha III subunit. The similarity in organization of these two ATPase genes and the homology in the primary structures of their proteins (approximately 60%) suggest that these two genes were derived from a common ancestral gene. However, the 5'-flanking regions of the genes for (H+ + K+)-ATPase and the (Na+ + K+)-ATPase alpha (+) subunit show no apparent sequence homology, indicating that their transcriptions are regulated differently. The control region of the fast-twitch sarcoplasmic reticulum Ca2(+)-ATPase gene also showed no sequence homology to that of (H+ + K+)-ATPase. The 5'-flanking region of the (H+ + K+)-ATPase gene contains potential binding sites for RNA polymerase II and various transcriptional regulation factors and several direct and inverted repeat sequences which may be important for specific and controlled expression of the gene in gastric parietal cells. There are two polyadenylation signals in the 3'-flanking region of the (H+ + K+)-ATPase gene, but the sequence of this region shows no homology to those of the corresponding regions of the genes for the (Na+ + K+)-ATPase alpha and alpha III subunits.
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PMID:Human gastric (H+ + K+)-ATPase gene. Similarity to (Na+ + K+)-ATPase genes in exon/intron organization but difference in control region. 216 Sep 52

The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
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PMID:Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. 216 40

Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates. A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis. The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template. Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II). The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble. The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.
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PMID:Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates. 216 70

Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures. A 100-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement tsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication.
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PMID:Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system. 217 Nov 87

We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.
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PMID:Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome. 217 87

By use of techniques described recently for lac permease [Roepe, P.D., & Kaback, H.R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6087], the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system. As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion. Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea. Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea. Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport. The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins.
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PMID:Isolation and functional reconstitution of soluble melibiose permease from Escherichia coli. 218 31

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.
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PMID:Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. 221 17

U6 small nuclear RNA (snRNA) is a required component in the splicing of eukaryotic pre-mRNAs. Mammalian U6 snRNA was synthesized in vitro by T7 RNA polymerase and purified on polyacrylamide gels. This U6 RNA, with pppG on its 5' end, was accurately capped to CH3-O-pppG, when incubated with HeLa cell extract and this capping was dependent on the capping signal present within the U6 snRNA. When gamma-32P-labeled U6 RNA was used as a substrate, the U6 cap formed in vitro retained this labeled gamma-phosphate, indicating that the cap formation involves the methylation of the gamma-phosphate incorporated during transcription. U6 snRNAs with ppG or pG as their 5' ends, were not capped in this in vitro capping system. Capping of U6 snRNA in vitro requires at least two components, a heat-labile component and S-adenosylmethionine as a methyl group donor. The data presented here show that capping of U6 snRNA can be uncoupled from transcription and that the mechanism of U6 snRNA cap formation differs markedly from the capping mechanism of mRNAs and other U snRNAs where capping is cotranscriptional. While many methyltransferases have been characterized earlier, this is the first report of a methyltransferase that is specific to phosphate residues. This in vitro capping system will be useful for purification and studies on the U6 snRNA sequence-dependent methyltransferase activity.
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PMID:Capping of U6 small nuclear RNA in vitro can be uncoupled from transcription. 234 79

The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.
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PMID:Role of the promoter in activation of transcription by nitrogen regulator I phosphate in Escherichia coli. 240 58


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