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 E. coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator. In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques. Under synchronized transcription initiation conditions in vitro, three closely-spaced transcription pause sites were identified. These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G112, G114 and A116 of the thr leader RNA. The results showed that the half-life of the thr paused complexes at G112 and G114 could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions. In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein. The thr leader complex that paused immediately before residues G112 and G114 of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents. The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites. However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein.
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PMID:Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex. 170 79

Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE-pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper-repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build-up of the full-length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.
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PMID:Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli. 171 Mar 13

We have developed a system for analysis of discrete steps in vaccinia virus early mRNA synthesis during a single round of transcription in vitro. A synthetic early promoter is used to direct transcription by vaccinia RNA polymerase of a G-less cassette in linear duplex DNA. Omission of GTP from transcription reactions leads to the formation of ternary elongation complexes paused stably at the end of the G-less cassette. These complexes can be induced to elongate by provision of GTP. While initiation of transcription is sensitive to low concentrations of salt and Sarkosyl, elongation is relatively resistant to these agents. Termination can be studied in a single synthetic cycle by forming transcription complexes paused just proximal to the termination signal TTTTTNT that can subsequently elongate and terminate. By selectively incorporating the termination-inhibiting analog BrUMP into proximal and distal portions of the nascent transcript, we localize the termination signal within or near the sequence UUUUUNU in the nascent RNA. We show that access of the vaccinia termination factor (VTF/capping enzyme) to the transcriptional apparatus can occur subsequent to initiation and synthesis of a 390-nucleotide nascent RNA. Termination is more sensitive to inhibition by salt and Sarkosyl than in elongation. This sensitivity is not reversed by preincubation of VTF with the transcription complex. Finally, we confirm the identity of VTF and vaccinia mRNA capping enzyme by demonstration of VTF activity associated with capping enzyme expressed in Escherichia coli.
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PMID:Discrete functional stages of vaccinia virus early transcription during a single round of RNA synthesis in vitro. 171 78

The effect of aflatoxin B1 (AFB1) on the template function for RNA synthesis of several single and double-stranded synthetic DNAs containing cytosine (C) and/or hypoxanthine (H) bases is studied in vitro. The results indicate that AFB1, after liver microsome activation, strongly inhibits the template function of poly[d(I-C)] and has little, if any, effect on polydI.polydC, polydI, or polydC. This conclusion is reached whether rat liver nuclear free RNA polymerase or E. coli RNA polymerase is used for the transcription. The mechanism of this inhibition is believed mainly due to the inhibition of elongation of RNA synthesis, because autoradiography of the [alpha-32 P]GTP labeled RNAs after polyacrylamide gel electrophoresis clearly shows that the size of the RNA from AFB1 treated group is dramatically reduced. The evidence that the selective inhibition of poly[d(I-C)] template function is a direct reflection of the binding of AFB1 to poly[d(I-C)] is provided by the use of radioactive [3H]AFB1 for the binding and by spectrum analysis of the appearance of a broad AFB1-DNA adduct peak between 300 nm and 400 nm right after the typical DNA peak at 260 nm. These data, which are in direct support to our recent report (F.L. Yu, et al., Carcinogenesis, 11, 475-478, 1990), suggest that the binding of AFB1 prefers alternating, double-stranded DNA, and the binding affinity of AFB1 to DNA is greatly reduced when the bases are in either single- or double-stranded homopolymer forms.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcriptional effect of aflatoxin B1 on cytosine and/or hypoxanthine containing DNAs. 171 92

The kinetics of formation of abortive initiation products during transcription of a synthetic template (encoding the transcript GAUGGC) by T7 RNA polymerase have been determined. This study revealed that while total RNA was formed in the reaction as expected, the levels of the dinucleoside tetraphosphate guanylyl-3',5'-adenosine-5'-triphosphate (pppGpA) and trinucleoside pentaphosphate guanylyl-3',5'-adenosine-3',5'-uridine-5'-triphosphate (pppGpApU) formed by premature termination of transcription reached a maximum after 10 min, and then decreased. Transcription of the same template, in the presence of either [gamma-32P]GTP and ATP, or GTP and [alpha-32P]ATP, gave the 32P-labeled dinucleotides *pppGpA and pppG*pA. Incorporation of each of these substrates into longer RNA transcripts in the same enzyme-template system was demonstrated. The incorporation was shown to require the presence of template in the reaction mixture. The requirement for base complementarity restricts the position of incorporation to that of initiating (5') nucleotide. Transcription of a second template, which encodes an RNA transcript having the partial sequence GpA at two internal positions, in the presence of each of the labeled dinucleoside tetraphosphates, failed to bring about the synthesis of significant yields of any longer radiolabeled transcripts. It is concluded that dinucleoside tetraphosphate (and perhaps trinucleoside pentaphosphate) can function as initiating nucleotides when complementary to the nucleotide sequence at promoter regions. However, a dinucleotide is not used as substrate for subsequent chain elongation in T7 RNA polymerase catalyzed transcription reactions.
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PMID:Abortive products as initiating nucleotides during transcription by T7 RNA polymerase. 171 17

Killer virions isolated from infected Saccharomyces cerevisiae cells contain an RNA polymerase activity which catalyzes the transcription in vitro of positive polarity RNAs from the L-A and M double-stranded RNA genomic segments of the virus. The RNA polymerase can initiate transcription in vitro with gamma-thio-GTP, whose thiophosphate group is found on the 5' terminus of transcripts. Transcripts produced in vitro by the virion-associated RNA polymerase in the presence of 7mGpppG are significantly more active as translational templates than are transcripts produced in its absence. However, unlike Escherichia coli RNA polymerase transcripts from viral cDNA made in the presence of 7mGpppG, transcripts produced by viral RNA polymerase in the presence of 7mGpppG fail to bind to antibody against 7mG.
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PMID:Yeast killer virus transcription initiation in vitro. 173 38

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (gamma-32P)ATP and (gamma-32P)GTP.
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PMID:Isolation and partial characterization of a protein kinase NII from wheat germ chromatin. 187 18

Four adjacent Leu codons within the leu leader RNA are critically important in transcription attenuation-mediated control of leu operon expression in Salmonella typhimurium and Escherichia coli (P. W. Carter, D. L. Weiss, H. L. Weith, and J. M. Calvo, J. Bacteriol. 162:943-949, 1985). The leader region from S. typhimurium was altered by site-directed mutagenesis to produce constructs having between one and seven adjacent Leu codons, all CUA. leu operon expression was measured in strains containing six of these constructs, each integrated into the chromosome in a single copy. Operon expression was sufficiently high that all strains grew in minimal medium unsupplemented by leucine. Expression of the operon was measured in strains cultured in such a way that their growth was limited by the intracellular concentration of either leucine or of leucyl-tRNA. In general, the leu operon for each construct responded similarly to the parent construct in terms of the degree of expression as a function of the degree of limitation. However, a strain containing (CUA)1 and, to a certain extent, a strain having (CUA)2 responded somewhat more sluggishly and strains containing (CUA)6 and (CUA)7 responded more sensitively to limitations than did the parent construct. In addition, DNA fragments containing the leu promoter and leader region were used as templates in in vitro transcription reactions employing purified RNA polymerase. With nucleoside triphosphate concentrations of 200 microM, RNA polymerase paused during transcription of the leu leader region at a site about 95 bp downstream from the site of transcription initiation. The halftimes of the pause were 1 min at 37 degrees C and 3 min at 22 degrees C. The pause was lengthened substantially when the GTP concentration was lowered to 20 micromoles. Our results are interpreted most easily in terms of an all-or-none model. Given two Leu control codons, the operon responds with nearly maximum output over a wide range of leucine limitation, and that outcome does not change much with increasing numbers of control codons.
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PMID:Transcription attenuation-mediated control of leu operon expression: influence of the number of Leu control codons. 199 84

The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot". 205 36

The diadenylate triphosphates ppp5'A2'p5'A and ppp5'A3'p5'A were found to inhibit the purified RNA polymerase ('nucleocapsid') complex from vesicular stomatitis virus (VSV). The corresponding diadenylate monophosphate p5'A2'p5'A did not inhibit, nor did the triadenylate triphosphate ppp5'A2'p5'A2'p5'A; the diadenylate diphosphate pp5'A2'p5'A had intermediate inhibitory activity. Increasing the concentration of ATP, GTP or CTP in the reaction mixture decreased inhibition by ppp5'A2'p5'A, while UTP had minimal or no protective effect. ppp5'A2'p5'A did not protect the RNA polymerase from inactivation by N-ethylmaleimide. This suggests that the action of ppp5'A2'p5'A occurs at a site on the enzyme that is distinct from the N-ethylmaleimide-protecting, ATP-binding site characterized previously.
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PMID:Inhibition of the RNA polymerase of vesicular stomatitis virus by ppp5'A2'p5'A and related compounds. 216 Jul 97


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