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)

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

The RNA-dependent RNA polymerase induced in BHK 21 cells by infection with foot-and-mouth disease virus has been isolated from the replication complex. It contains a major, virus-coded protein with mol. wt. 56 000 which appears from serological studies and tryptic peptide mapping to be the same as the virus infection associated (VIA) antigen and the protein P56 found in cells infected with the virus. Other virus coded proteins and a host cell protein were present in the partially purified replication complex but were removed by digestion with ribonuclease T1, leaving only the major virus coded protein. The tryptic peptide maps of the VIA antigen of the seven serotypes of the virus were similar, suggesting a high level of conservation in that region of the genome coding for the RNA polymerase of each type.
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PMID:Purification and identification of the RNA-dependent RNA polymerase of foot-and-mouth disease virus. 23 34

When treated at pH less than 4.5, yeast nuclei or chromatin lose endogenous RNA synthetic activity. This activity is regained by addition of exogenous RNA polymerases. The specificity of transcription in this system by homologous RNA polymerases I and III has been investigated by gel electrophoresis, hybridization analysis, and RNase T1 mapping. Exogenous RNA polymerase I selectively transcribes rRNA genes. The transcription of these genes by polymerase I is 30- and 8-fold more selective than RNA polymerase III and Escherichia coli polymerase holoenzyme, respectively. Exogenous RNA polymerase III synthesized RNAs similar in size to authentic 5 S RNA, 4.5 S pre-tRNA, and 4 S tRNA. Eleven per cent of this RNA is 5 S RNA as determined by hybridization. Neither polymerase I nor E. coli polymerase synthesizes detectable quantities of RNA in this size range. AT1 ribonuclease digestion of 5 S RNA synthesized by exogenous RNA polymerase III acting on acid-treated chromatin gives a fragment pattern corresponding to that of 5 S RNA. Thus, RNA polymerase III transcribes the entire 5 S gene in this system.
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PMID:Specific gene transcription in yeast nuclei and chromatin by added homologous RNA polymerases I and II. 36 64

RNA was synthesized in vitro from a T3 DNA template by T3 RNA polymerase and subsequently separated into seven discrete size classes (molecular weights ranging between 0.21 x 10(6) and 6.2 x 10(6)) by electrophoresis in polyacrylamide slab gels. RNase T1-generated 3'-terminal oligonucleotide fragments were then selectively isolated from either the unfractionated total RNA or the gel-purified specific transcripts by chromatography on columns of dihydroxyboryl-cellulose. Sequence analysis of these oligonucleotide products indicated that the unfractionated transcripts as well as all the individual major RNA species examined had a unique sequence, (Gp)UpUpUpUpUpGOH, at their 3' termini. The specificity of this sequence, as well as the total lack of any sequence heterogeneity at the ends of these transcripts, indicates a high degree of specificity of termination during transcription in this system.
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PMID:Termination of transcription by bacteriophage T3 RNA polymerase: homogeneous 3'-terminal oligonucleotide sequence of in vitro T3 RNA polymerase transcripts. 38 30

Long pyrimidine tracts, purified from Drosophila melanogaster DNA after treatment with formic acid-diphenylamine, were used as template for E. coli RNA polymerase to produce a polynucleotide containing only purines. This polypurine RNA hybridized specifically to D. melanogaster DNA with high efficiency at low Cot values. The resulting hybrid showed high thermal stability. When polypurine RNA was subjected to complete hydrolysis with ribonuclease T1, over 90% of the nucleotide products were ApGp and ApApGp. Partial hydrolysis yielded a distinct additional component, ApApGpApGp + ApGpApApGp. We conclude that the major sequence in the polypurine transcript is (ApGpApApGp)n. In situ hybridization to salivary gland polytene chromosomes and to metaphase chromosomes from neural ganglia indicated that polypyrimidines complementary to polypurine RNA are located in heterochromatin. In femal cells, the predominant labeling was on centromeric heterochromatin of the 2nd chromosome. We have verified the location of polypyrimidines in neural ganglion cells, by using a cytological marker of chromosomes 2. In male cells, hybrid was also found on the Y chromosome.
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PMID:Polypyrimidine segments in Drosophila melanogaster DNA: II. Chromosome location and nucleotide sequence. 80 50

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.
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PMID:Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome. 131 81

The Saccharomyces cerevisiae transcription factors (TF) IIIB and IIIC assemble onto their respective DNA-binding sites on the SUP4 tRNA(Tyr) gene at 0 degrees C. RNA polymerase III specifically associates at 0 degrees C with this TFIIIC-TFIIIB-DNA complex to form a stable "closed" promoter complex in which the DNA surrounding the transcriptional start retains its duplex form. Promoter "opening" is a temperature-dependent and readily reversible process that involves up to 22 unwound base-pairs of DNA, and can be followed by analyzing the hyperreactivity of thymine to KMnO4 oxidation. This promoter opening increases progressively from 10 degrees C to 40 degrees C, with at least two regions within the transcription bubble appearing to melt independently. In contrast, the temperature dependence of forming an initiated transcription complex containing a 17 nucleotide nascent RNA chain displays a sharp transition between 10 degrees C and 15 degrees C. When RNA polymerase initiates transcription under conditions that limit the nascent RNA chain to less than six nucleotides, there is no displacement of the transcription bubble. These transcription complexes are distinguishable from "open" promoter complexes in their maintenance of the transcription bubble at 0 degrees C, and from transcription complexes with more extended (17 nucleotide) RNA chains in their sensitivity to disruption by heparin. In light of recent results by others that demonstrate a requirement for an RNA transcription factor in a Bombyx mori-based in vitro RNA polymerase III transcription system, we have searched for a comparable component in the S. cerevisiae-derived system. We show that if an RNA component is required in the yeast-derived system, it is not susceptible to inactivation by massive amounts of micrococcal nuclease, RNase A, or RNase T1.
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PMID:Formation of open and elongating transcription complexes by RNA polymerase III. 161 62

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
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PMID:Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex. 244 22


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