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)

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.
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PMID:Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. 266 28

The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure that contains four essential single-stranded nucleotides. Additional specificity is thought to be due to four or five ionic contacts between the protein and phosphates on the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5'-O-(1-thiotriphosphates) [NTP(alpha S)s], allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site were synthesized with different NTP(alpha S)s and tested for coat protein binding to deduce positions of thiophosphates that alter the binding affinity. Of the twenty-one phosphate positions in the molecule, two were found to decrease the Ka 3-fold when substituted with a thiophosphate, one position decreased the Ka 10-fold, and one position increased the Ka 10-fold. Substitution of any of the other 17 positions with thiophosphates does not alter the Ka. The four positions that alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates affect binding because they contact coat protein directly.
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PMID:Determination of RNA-protein contacts using thiophosphate substitutions. 266 62

Lac permease, a polytopic membrane protein from Escherichia coli, has been purified in soluble form by overexpressing the lacY gene by means of the T7 RNA polymerase system. Soluble permease is dissociated from membranes with urea or other chaotropes and appears after the membrane is saturated with newly synthesized permease. Remarkably, this form of the permease appears to remain soluble in phosphate buffer at neutral pH after removal of urea, although it aggregates in a time- and concentration-dependent manner. Importantly, soluble permease behaves as a monomer during size-exclusion chromatography with or without urea, contains less than 3 mol of organic phosphate per mol of protein, and is largely helical. Soluble permease binds p-nitrophenyl alpha-D-galactopyranoside approximately 40% as well as permease in the native environment of the membrane and can be reconstituted into phospholipid vesicles that catalyze lactose counterflow or active transport in response to a membrane potential (interior negative). The results suggest that lac permease can assume a nondenatured conformation in aqueous solution.
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PMID:Characterization and functional reconstitution of a soluble form of the hydrophobic membrane protein lac permease from Escherichia coli. 266 55

The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a specific inhibitor for RNA polymerase II transcription in vivo and in vitro [Tamm + Sehgal (1978) Adv. Virus Res. 22, 187-258; Zandomeni & Weinmann (1984) J. Biol. Chem. 259, 14804-14811]. The effect on RNA polymerase II-specific transcription seems to be mediated by its inhibition of nuclear casein kinase II [Zandomeni, Carrera-Zandomeni, Shugar & Weinmann (1986) J. Biol. Chem. 261, 3414-3419]. Inhibition studies indicated that DRB acted as a mixed-type inhibitor with respect to casein and as a competitive inhibitor with respect to the nucleotide phosphate donor substrates. The DRB inhibition constant is 7 microM for the calf thymus casein kinase II, with regard to both ATP and GTP.
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PMID:Kinetics of inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on calf thymus casein kinase II. 280 63

U6 small nuclear RNA (snRNA), a component of eukaryotic spliceosomes, is required for splicing of nuclear pre-mRNAs. Whereas trimethylguanosine cap-containing U sn-RNAs are transcribed by RNA polymerase II, the U6 RNA is transcribed by RNA polymerase III and contains a nonnucleotide cap structure on its 5' end. We characterized the cap structure of human U6 snRNA and show that the gamma phosphate of the 5' guanosine triphosphate is methylated. The mobilities of in vivo-modified gamma phosphate from the 5' end of HeLa U6 RNA were identical to the synthetic monomethyl phosphate (CH3-O-P) in two-dimensional chromatography and two-dimensional electrophoresis. The cap structure of U6 RNA is distinct from all other cap structures characterized thus far.
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PMID:Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA. 281 91

The murine urokinase-type plasminogen activator (uPA) gene has been isolated from a BALB/c liver DNA cosmid library and its nucleotide sequence established. The gene is organized into 11 exons comprising 34.7% of the 6710 base pair (bp) region spanning the interval between the presumed transcription initiation and polyadenylation sites. The transcription initiation site is flanked by common RNA polymerase II promoter elements, including a TATA box and a potential transcription factor Sp1 binding site. A large polypurine tract of the structure (AG)22(AGGG)16(AG)28 is located 79 bp upstream of the 5'-terminus. It was highly sensitive to the single-strand-specific nuclease S1, suggesting a non-B-DNA conformation of unknown significance. Consistent with the well-documented influence of adenosine cyclic 3',5'-phosphate (cAMP) on uPA gene expression, there is a dodecanucleotide homologous to proposed regulatory sequences identified in other cAMP-modulated genes. Comparison of the murine uPA gene to the previously described porcine and human uPA genes revealed an unusually high degree of evolutionary (interspecies) sequence conservation that was not limited to exons but included introns and flanking sequences as well.
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PMID:The murine urokinase-type plasminogen activator gene. 283 40

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.
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PMID:Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors. 284 29

The intracellular killer virions of yeast co-purify with an RNA polymerase activity which catalyzes the synthesis of full-length transcripts of the two viral genomic double-stranded RNA segments. This polymerase utilizes ribonucleoside diphosphates or triphosphates as substrates. The virions have other associated nucleotide-metabolizing enzyme activities, including nucleoside diphosphate kinase, adenosine monophosphate kinase, and nucleoside triphosphate phosphotransferase, an activity which catalyzes the exchange of gamma-phosphate from any ribonucleoside triphosphate with any ribonucleoside or deoxyribonucleoside triphosphate. The purified virions also contain an inorganic pyrophosphatase activity. These enzymes may allow the virus to utilize nucleotide pools distinct from those utilized in host cell transcription.
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PMID:Nucleotide phosphotransferase, nucleotide kinase and inorganic pyrophosphatase activities of killer virions of yeast. 284 57

Escherichia coli rpoN mutants lack sigma 54 and are therefore unable to initiate the transcription of glnA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase. We have found that the dependence on sigma 54 can be overcome by mutations that have apparently created a new sigma 70-dependent promoter. The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2. The initiation of transcription at the new promoter is inhibited by sigma 54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2. The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, sigma 54-RNA polymerase is bound at glnAp2.
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PMID:Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli. 288 48

The biosynthesis and phosphorylation of nuclear proteins of thymocytes were investigated in rats after 4.0 Gy whole-body X-irradiation during the period which precedes DNA degradation in lymphoid cells. Proteins were separated by two-dimensional gel-electrophoresis, detected by Coomassie blue staining. Incorporation of [35S]methionine into proteins and phosphorylation of proteins with [32P]inorganic phosphate were determined by scanning densitometry of autofluorograms and autoradiograms, respectively. No change in the quantity of proteins was observed 1 h after irradiation. Decrease in specific activity of [35S]methionine-labelled proteins was seen in most protein fractions. Significant enhancement of phosphorylation of three proteins was established, characterized by molecular weight and pH: MW 20 kD, pH 6.8; MW 35 kD, pH 5.8 and MW 48 kD, pH 5.8. These results suggest that immediately after X-irradiation a short-term increase of chromatin-bound non-histone protein phosphorylation occurs. This finding, along with the previously described enhancement of RNA polymerase II in thymocytes (Zhivotovsky et al. 1982) suggests a temporary gene activation shortly after X-irradiation of the rat.
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PMID:Nuclear protein synthesis in thymocytes of X-irradiated rats. 290 95


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