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 analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.
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PMID:Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. 246 38

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.
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PMID:Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome. 247 95

Reverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA-RNA compound. In this compound a single-stranded DNA is connected to RNA at a specific G residue by a 2'-5' phosphodiester linkage. The precursor for the DNA-RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA-RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA-RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.
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PMID:Reverse transcriptase in bacteria. 248

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52-kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52-kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52-kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20,000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric. The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N-ethylmaleimide, and exhibits a pH optimum of 9.0-9.5. The enzyme releases oligoribonucleotides with 3'-OH and 5'-phosphate ends, probably in an exonucleolytical manner. The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431-433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.
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PMID:Serological analysis and characterization of calf thymus ribonuclease H IIb. 255 85

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.
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PMID:Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H. 255 16

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.
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PMID:In vitro assembly of yeast U6 snRNP: a functional assay. 256 Jul 55

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site-directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion-exchange HPLC, yielded a pure preparation of a 3'-terminal, 112-nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T7 RNA polymerase, resulted in the isolation of an 88-nucleotide fragment. Finally, a 44-nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy. Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44-nucleotide fragment. Also the analogue of the T-stem and T-loop of the tRNA-like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+-containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species. To obtain direct biophysical evidence for base-pairing and stacking interactions in the pseudoknot, NMR studies were initiated. The first proton-NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112-nucleotide and 88-nucleotide fragment are too complicated to be solved at present. In the case of the 44-nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.
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PMID:Biochemical and biophysical analysis of pseudoknot-containing RNA fragments. Melting studies and NMR spectroscopy. 277 53

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.
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PMID:Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. 283 68

I have previously reported an activity in HeLa cells which facilitates transcript displacement by purified mammalian RNA polymerase II in vitro. I have shown that this activity copurifies with one of two separable ribonuclease (RNase) H activities in HeLa cells. The RNase H activity in question has characteristics similar to those reported for RNase H2b from calf thymus. RNase H proteins purified from several other sources including Escherichia coli also show renaturase activity. When the renaturase/RNase H protein is present during transcription by purified RNA polymerase II, transcripts are truncated close to the 5' end, and the remainder of the transcript is displaced normally from its template by the polymerase. Since RNA polymerase II dependent transcripts in vivo normally require the presence of the 5'-triphosphate terminus for capping, the in vivo significance of RNase H as a renaturase factor is presently not understood. However, the in vitro action of renaturase/RNase H suggests that the mechanism of this reaction may involve R-loop displacement after formation of a short single-stranded region of DNA on the template strand following hydrolysis of a hybrid transcript oligonucleotide by RNase H.
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PMID:Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro. 283 25

The replication of plasmid pBR322 DNA has been reconstituted with purified proteins from Escherichia coli. Initiation of the leading-strand requires RNA polymerase holoenzyme, DNA polymerase I, RNase H, and DNA gyrase. Initiation of the lagging-strand requires the primosomal proteins (the dnaB, dnaC, and dnaG proteins, replication factor Y (protein n') and proteins i, n, and n") and the single-stranded DNA binding protein. DNA polymerase III holoenzyme is required for extensive elongation of the nascent DNA chains. The products of this replication reaction are primarily nonsegregated daughter molecules. However, the addition of small amounts of soluble extract from E. coli results in the completion and segregation of these molecules to give mature form I DNA, suggesting that additional factors are required for this process. Topoisomerase I is necessary to make the replication system specific for pBR322 DNA as a template, indicating that the linking number of the DNA, determined by an equilibrium between the opposing activities of topoisomerase I and DNA gyrase, plays a crucial role in determining the reactivity of the DNA molecule toward initiating DNA replication. The function of the proteins involved in the replication of this closed-circular, double-stranded, superhelical DNA is discussed.
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PMID:Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity. 299 Dec 40


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