Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.
Nucleic Acids Res 1979 Dec 11
PMID:The nucleotide sequence at the 5' end of foot and mouth disease virus RNA. 23 62

Using BspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain. The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant RTs were assayed. The elucidation of the structure of HIV-1 RT makes it possible to determine the locations of specific mutations in the three-dimensional structure of HIV-1 RT [E. Arnold, A. Jacobo-Molina, R. G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature (London) 357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T. A. Steitz, Science 256:1783-1790, 1992]. The mutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase activity without significantly affecting RNase H activity.
J Virol 1992 Dec
PMID:Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase. 127 5

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
J Biol Chem 1992 Dec 25
PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
Biochem Biophys Res Commun 1992 Dec 15
PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).
Experientia 1992 Dec 01
PMID:Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843. 128 6

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.
Protein Expr Purif 1992 Dec
PMID:Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product. 128 95

The element; Ty4 is a retrotransposon present in low copy number in the genome of Saccharomyces cerevisiae [Stucka et al., Nucleic Acids Res. 17 (1989) 4993-5001]. We have determined the complete nucleotide sequence of one such element from a particular strain and compared it to the other two elements occurring in this strain. The genomic organization of Ty4 is homologous to that found in other retrotransposons of the Ty1-copia group. The internal part of the element contains two large open reading frames (TY4A and TY4B) overlapping by 226 bp in a + 1 mode. TY4A reveals characteristics of the gag portion of retrotransposons and retroviruses, while TY4B consists of a protease, an integrase, a reverse transcriptase, and an RNase H domain (in that order). Our analyses suggest that only one of these copies might be transpositionally active. Sequence comparisons at the amino acid level show that the domains in Ty4 diverge considerably from those of other retrotransposons. The greatest similarity is seen between the reverse transcriptases (50%), the proteases (40%), and the integrases (30%) of Ty4, Ty1/2 and copia, respectively, whereas the degree of similarity for all other entities of these elements is much lower. Considering evolutionary aspects of the retrotransposons, we have to conclude that Ty4 has diverged at an early stage from the progenitors of other known retroelements and represents a novel and independent subgroup of the Ty1-copia class of retrotransposons.
Gene 1992 Dec 01
PMID:Molecular analysis of the yeast Ty4 element: homology with Ty1, copia, and plant retrotransposons. 133 37

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboxylase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboxylase synthesis in response to mRNA from D-R L1210 cells was brought about by 5'-AAAGCTGCTCATGGTTCT-3' which is complementary to the sequence from -6 to +12 of the mRNA sequence but there was no inhibition by 5'-TGCAGCTTCCATCACCGT-3'. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from -6 to +12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.
Mol Cell Biochem 1992 Dec 16
PMID:Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides. 133 19

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.
EMBO J 1992 Dec
PMID:A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. 146 25

The 3'-terminal sequences of flavivirus genomes within approx. 100 nucleotides (nt) have been suggested to have a highly conserved secondary structure, as based on the known nt sequence data and free-energy calculations using computer programs. To test the existence of a secondary structure in solution, we devised a strategy to generate truncated RNA molecules from about 0.3-1.4 kb in length, having the same polarity and nt sequence as dengue virus type 2 (DEN-2) RNA (New Guinea-C strain). When these labeled RNA molecules were digested by RNase A, and analyzed by denaturing polyacrylamide-gel electrophoresis, three resistant fragments of 16, 20 and 23 nt in length were reproducibly obtained. To examine whether these RNase A-resistant (RNaseR) fragments emerged from a stable secondary structure formed in solution consisting of 3'-terminal sequences, hybridization of the RNaseR fragments to four chemically synthesized oligodeoxyribonucleotides (oligos), complementary to nt 1-24, 25-48, 49-72, and 73-96 from the 3' terminus of DEN-2 RNA, followed by RNase H digestion were carried out. Oligos complementary to nt 25-48 and 49-72 from the 3' end of DEN-2 RNA were sufficient to render all three RNaseR fragments susceptible to RNase H digestion. These data indicate that a stable secondary structure is formed in solution involving nt 18-67 from the 3' terminus. The potential use of these unique transcripts to identify the viral and/or host proteins which might interact at the 3' terminus of DEN-2 RNA during initiation of replication is discussed.
Gene 1991 Dec 15
PMID:Detection of stable secondary structure at the 3' terminus of dengue virus type 2 RNA. 166 Aug 36


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