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)

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.
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PMID:Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition. 14 Jan 66

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.
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PMID:T7 gene 6 exonuclease has an RNase H activity. 36 24

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.
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PMID:Identification of two different RNase H activities associated with yeast RNA polymerase A. 38 60

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay. Gel retardation assays revealed that both full-length tRNA species, as well as a 5'-truncated h-tRNA(UUULys3) molecule containing 24 nucleotides (nt) at the 3' end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Competition studies with these three tRNA species demonstrate that the 3' end of h-tRNA(UUULys3) contributes to the interaction with HIV-1 RT. Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) were also able to interact with the enzyme, whereas unrelated RNA molecules such as E. coli 5S rRNA did not bind to RT. Both b-tRNA(SUULys3) and h-tRNA(UUULys3) molecules, as well as the 5'-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process. E. coli tRNA(UUULys) and tRNA(UUCGlu2), although able to interact with HIV-1 RT, failed to prime retroviral transcription. Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3' ends of h-tRNA(UUULys3) and b-tRNA(SUULys3) are still present in the cDNA product, whereas the 5' ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.
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PMID:Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis. 137 59

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
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PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The presence of such unusual structures is confirmed by the following evidence. When a preparation of matrix DNA is electrophoresed in agarose an RNA component always migrates together with DNA. There should be a close interaction between DNA and RNA in such a preparation because the presence of the RNA component causes resistance of DNA to DNase I and Exo III. An intimate, hybrid-type association of part of the RNA component with DNA is indicated also by the fact that about 20% of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis with alpha-amanitin and actinomycin D it was shown that the bulk of associated RNA is transcribed by RNA polymerase III. Hybridization experiments showed similarity between the DNA sequences isolated from the complexes and those from the base of dehistonized DNA loops obtained by high-salt extraction of nuclei. This observation suggests that the complexes might represent attachment sites of nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA component. Experiments with induction of erythroid differentiation indicated that a profound reorganization of the nucleus, accompanying terminal differentiation, leads to a striking reduction in the number of complexes and thus in the number of attachment sites. This suggests that the complexes should function as transient attachment sites.
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PMID:DNA-RNA complexes that might represent transient attachment sites of nuclear DNA to the matrix. 169 80

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.
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PMID:HIV-1 reverse transcriptase: structure predictions for the polymerase domain. 170 98


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