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)

The rational design of antiviral agents targeting the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) would greatly benefit from a more intimate knowledge of the structure of RT. Until now, the degree of sequence similarity between RT and E. coli DNA polymerase I (Pol I) has been thought to be confined to several small regions, suggesting little basis for homology molecular modeling. However, we have found that a region in the C terminal of the RT polymerase domain is homologous to a central region of Pol I that lies between the universal polymerase motifs A and C (specifically, helices N-O-P of the Pol I crystal structure); a single transposition closely aligns the RT and Pol I genes, revealing a similar domain structure with 20% residue identity, as well as the possible structural correlates of several RNA-dependent polymerase motifs. The RT from Myxococcus xanthus (a bacterium believed to have diverged from other species 2 billion years ago), if similarly transposed, shows homology to both HIV-1 and E. coli, suggesting the possibility of a very ancient divergence between the RT and Pol I polymerase genes. A second even more significant match to this E. coli region was found in the retroviral ribonuclease H (RNase H) domain, and corresponds precisely to a region that has been aligned by previous investigators with the E. coli RNase H, suggesting that Pol I helices O and P are homologous to helices A and D of the RNase H crystal structure, respectively. These results are consistent with a modular theory of molecular evolution.
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PMID:A transposition of the reverse transcriptase gene reveals unexpected structural homology to E. coli DNA polymerase I. 172 84

A new model of secondary and tertiary structure of higher plant 5S rRNA is proposed. It consists of three domains. Domain alpha includes stem I and loop A; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. We propose that the domains beta and gamma adopt RNA-A like structure due to irregularities caused by the different in size internal loops B and E and the bulges occurring in the model. A suggested bending of RNA could bring single stranded fragments of domains beta and gamma close enough to each other to allow tertiary interactions. The new model of plant 5S rRNA differs from those suggested previously for eukaryotic 5S rRNA, by arrangement of the domains beta and gamma and the base pairing scheme of domain gamma. The model is based on our results of partial digestion obtained with single and double strand specific nucleases. The experimental results were confirmed by computer aided secondary structure prediction analysis of all higher plant 5S rRNAs and computer modeling using energy minimalization approach. Further support of our model have been provided by experiments including alpha sarcin, ribonuclease H and chemical modifications.
Acta Biochim Pol 1990
PMID:Tertiary structure and computer modeling of plant 5S ribosomal RNA. 208 25

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
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PMID:Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. 245 55

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.
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PMID:HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. 247 33

Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase. These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells. In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity. The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.
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PMID:Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase. 618 37

The effects of point mutations of the conserved Asp443, Glu478, Asn494, and Asp498 residues in the RNase H domain of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) have been analyzed. The mutants fell into two classes: (i) functional RT, but not detectable ribonuclease H activity, and (ii) uncharacterizable phenotype due to protein instability in the context of the RT/protease Escherichia coli co-expression system (Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A., and Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358). The only mutation in the former class was D443A, whereas those in the latter included D443E, E478D, E478Q, D498E, D443A/D498N, D443E/D498N, D443Q/D498N, N494A, N494D, and N494Q. The results were interpreted in terms of the x-ray crystal structure of the HIV-1 RNase H domain (Davies, J. F., II, Hostomaska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 252, 88-95) and a general acid-general base hydrolysis mechanism (Katayanagi, K., Okumura, M., and Morikawa, K. (1993) Proteins Struct. Funct. Genet. 17, 337-346). The data suggested that structural perturbations within the RNase H domain interfered with maturation of the pol precursor by HIV-1 protease. Analysis of selected D443/D498 double mutants suggested that the destabilization caused by the D498N mutation could be suppressed by the formation of a new hydrogen bond between Asn498 and Asn443.
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PMID:Mutagenesis of the conserved aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498 residues in the ribonuclease H domain of p66/p51 human immunodeficiency virus type I reverse transcriptase. Expression and biochemical analysis. 751 54

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.
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PMID:Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli. 768 63

The polymerase activity of the p51 homodimeric form of HIV reverse transcriptase was characterized by activity gel analysis, steady-state kinetic measurements, and processivity assays, and the activity was shown to be highly similar to that for the p66/p51 heterodimer. Recombinant 51- and 66-kDa reverse transcriptase proteins were individually expressed from an HIV-1 Pol gene having an accumulation of natural amino acid mutations compared to the BH10 clone (Ratner et al., 1985). The preparation of an active p51 homodimer critically depended on low temperature during its expression in bacterial cultures. Activity gel analysis demonstrates that refolded p51 protein derived from denatured p66/p51 heterodimer yields an active polymerase. The p51 homodimer has approximately one-half the activity and processivity of the heterodimer, while both enzymes have similar thermostability. Steady-state measurements reveal no significant differences in apparent affinities for substrate or homopolymeric template-primer, suggesting that the subunits in both enzyme forms have similar conformations. Template challenge experiments show that the off-rates for template-primer are lower, but as indicated by primer extension analyses, processivity is less for p51 homodimer. These results show that the RNase H domain is not essential for the assembly of the functional polymerase, but suggest that it enhances processivity.
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PMID:HIV-1 reverse transcriptase: polymerization properties of the p51 homodimer compared to the p66/p51 heterodimer. 769 Nov 76

N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.
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PMID:Identification of proteolytic processing sites within the Gag and Pol polyproteins of feline immunodeficiency virus. 838 14

It has been reported recently that the human foamy virus (HFV) Pol polyprotein of 120 kDa is synthesized in the absence of the active HFV aspartic protease. To gain more information on how the 120-kDa Pro-Pol protein is synthesized, mutant HFV genomes were constructed and the resulting proviruses were analyzed with respect to HFV pol expression and infectivity. HFV proviruses that contain termination codons in the nucleocapsid domain of gag and thus lack a gag-pol overlap region assumed to be required for translational frameshifting, nevertheless expressed the 120-kDa Pro-Pol precursor, the 80-kDa reverse transcriptase/RNase H, and a 40-kDa integrase in amounts similar to those observed for wild-type genomes. Since a Gag-independent expression of authentic Pol proteins was detectable in cells transfected with eukaryotic HFV pol expression plasmids, the data indicate that the HFV Pol precursor of 120 kDa is expressed independently of Gag by a mechanism that does not rely on ribosomal frameshifting, since the postulated HFV Gag-Pol protein of 190 kDa was not detectable under the conditions used. Furthermore, replacement of the Met residue by Thr at position 9 in pol within the gag-pol overlap region resulted in strongly reduced HFV Pol polyprotein expression and infectivity of the resulting proviruses. This Met residue of pol conserved in foamy virus sequences is the likely candidate for translational initiation of the 120-kDa Pro-Pol polyprotein. trans complementation of the HFV mutant with the Met-to-Thr substitution in the pol gene by a eukaryotic plasmid that expressed the HFV Pro-Pol protein resulted in partial recovery of infectivity. When HFV pol was fused in frame to gag, an engineered 190-kDa Gag-Pol fusion protein was formed and the enzymatic activity of the HFV protease was partially retained. The results imply that HFV is the first retrovirus that expresses a Pol polyprotein without formation of a Gag-Pol fusion protein.
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PMID:The human foamy virus pol gene is expressed as a Pro-Pol polyprotein and not as a Gag-Pol fusion protein. 855 61


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