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)

A primase-reverse-transcriptase of Halobacterium halobium was purified by column chromatography on DEAE-cellulose, hydroxyapatite and carboxymethyl-cellulose, followed by sedimentation on a glycerol gradient. The enzyme is a multifunctional enzyme containing reverse transcriptase. DNA polymerase and RNase H activities and does not require a performed primer to initiate DNA synthesis. Using a single-stranded DNA as template, this enzyme synthesizes oligonucleotides (8-12 bases) that can be used a primer by Escherichia coli DNA nucleotidyltransferase I (DNA polymerase I, Klenow fragment). Two polypeptides of 67 and 57 kDa were found after 14750-fold purification of the enzyme.
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PMID:Reverse transcriptase in archaebacteria. Purification and characterization of a primase-reverse-transcriptase complex from Halobacterium halobium. 170 56

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.
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PMID:Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. 171 88

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

Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).
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PMID:NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. 172 72

We have purified from whole cell extracts of Saccharomyces cerevisiae a protein which alters the elongation properties of yeast RNA polymerase II in vitro. The yeast elongation stimulatory activity, YES, correlates with a 116-kDa protein and acts on both yeast and Drosophila RNA polymerase II during transcription of double-stranded dC-tailed templates. The stimulatory activity is specific for RNA polymerase II since it has no significant effect on the elongation properties of yeast RNA polymerase I or yeast RNA polymerase III. Elongation by RNA polymerase II can be stimulated by RNase H on dC-tailed templates; however, the stimulatory activity of YES is not due to RNase H activity. YES does not stimulate RNA polymerase II in the presence of manganese ions and therefore is distinct from the smaller elongation factor, S-II or DmS-II. YES is most similar to Drosophila factor 5 (mammalian TFIIF, or RAP30/74), an initiation factor that is also able to increase the rate of elongation of RNA polymerase II.
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PMID:Identification and purification of a yeast protein that affects elongation by RNA polymerase II. 185 Nov 72

We describe a scheme for isolation of new classes of mutants in the cell cycle of Escherichia coli. The mutants were selected as resistant to camphor vapors, which results in increased ploidy, and were subsequently screened for an increase in cell density and an increase in the gene dosage of the lac operon. Our mutations are located at four different places in the chromosome; we have named these loci mbr (moth ball resistant). mbrA maps to 68 min on the E. coli chromosome, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD to 90 min. mbrD mutations may be alleles of rpoB (a subunit of RNA polymerase). In addition to the selected or screened phenotypes, most of the mutants fail to grow on rich media or at high temperatures. We have examined the nine mutants under nonpermissive conditions, using several techniques to determine the cause of death. We have also coupled our mutations with lesions in dnaA, which is required for cell-cycle-specific DNA replication, and rnh (the gene for RNase H), which is required for specificity in the DNA initiation reaction, and determined the effects of the double and triple mutants under permissive and nonpermissive conditions. These tests have shown that bacteria mutated at mbrA do not tolerate a null mutation in rnh, indicating that they are dependent on DNA replication initiating at oriC. In contrast, mutations at mbrB, mbrC, and mbrD exhibit their phenotypes independent of oriC initiation of DNA replication, suggesting that the mutations affect factors that influence the DNA/cell ratio regardless of the origin of DNA replication. Based on our results, the mbr mutations appear to have defects in cell-cycle timing and/or defects in chromosomal partitioning.
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PMID:On the bacterial cell cycle: Escherichia coli mutants with altered ploidy. 217 33

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.
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PMID:Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. 230 48

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.
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PMID:Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. 240 71

RNA polymerase II will efficiently initiate transcription on linear duplex DNA which has been extended at its 3' ends by the addition of short stretches of polydeoxycytidine (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295). We have used such dC-tailed templates to identify factors affecting elongation by Drosophila RNA polymerase II (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). While studying these factors we have observed two unexpected characteristics of transcription of the tailed templates. First, we found that RNA polymerase II encountered a strong pause site after the incorporation of 14 nucleotides. This pausing was observed on all templates examined and with RNA polymerase II from a variety of sources. In addition, we found that ammonium ions markedly stimulated the polymerase, increasing both the efficiency with which the enzyme left the 14 base pause site and the subsequent rate of elongation. A factor previously shown to affect transcription of dC-tailed templates (factor 4, Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255) was found to cause transcript displacement and to stimulate the elongation rate approximately 2-fold. This factor copurified with an RNase H activity, and a model is presented for the mechanism of transcript displacement by RNase H. The observations presented here form a basis for further analysis of RNA polymerase II elongation and its modulation by transcription factors. They should also aid in the interpretation of other experiments in which dC-tailed templates are used.
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PMID:Elongation by Drosophila RNA polymerase II. Transcription of 3'-extended DNA templates. 245 24


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