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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hybrid nucleases consisting of an oligonucleotide fused to a unique site on the relatively nonspecific phosphodiesterase staphylococcal nuclease have been shown to sequence specifically cleave DNA. We have introduced mutations into the binding pocket of the nuclease which lower the kcat/Km of the enzyme. Hybrid nucleases generated from these mutants sequence selectively hydrolyze single-stranded DNA in a catalytic fashion, and under a much wider range of conditions than was previously possible. One such hybrid nuclease (Y113A, K116C) was able to site selectively cleave single-stranded M13mp7 DNA (7214 nt), primarily at one phosphodiester bond. Another hybrid nuclease (Y113A, L37A, K116C) catalyzed the hydrolysis of a 78-nt DNA substrate with a kcat of 1.2 min-1 and a Km of 120 nM. The effects of variations in the length and sequence of the oligonucleotide binding region were examined, as were changes in the length of the tether between the oligonucleotide and the enzyme. Cleavage specificity was also assayed as a function of substrate DNA primary and secondary structure and added poly(dA).
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PMID:Generation of a catalytic sequence-specific hybrid DNase. 260 84

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.
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PMID:Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. 368 96

A 1.6-kilobase-pair DNA fragment derived from the Escherichia coli chromosome was analyzed by Tn3 transposon insertion and deletion mapping to locate a mutator gene, dnaQ (mutD), and the rnh gene that codes for RNase H. When a strong promoter, PL of lambda phage, was placed at the right- and left-side of the cloned DNA fragment, the dnaQ protein and RNase H, respectively were overproduced. These results suggested that the two genes are transcribed in opposite directions and that their promoters are located in a narrow region between the genes. Nucleotide sequence analysis confirmed this and further revealed that transcriptional and translational initiation signals for the two genes overlap. From the sequence data it was deduced that the dnaQ protein and RNase H consist of 243 and 155 triplets and have molecular weights of 27,500 and 17,500, respectively. dnaQ81 amber mutant showed two codon alterations, CAG(glutamine-195) leads to TAG(amber) and ACA(threonine-193) leads to ATA(isoleucine). The dnaQ-lacZ and the rnh-lacZ fused genes were constructed and hybrid proteins with beta-galactosidase activity were produced. From beta-galactosidase levels it was estimated that the promoter for dnaQ is 5 times more active than that for rnh.
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PMID:Structure and expression of the dnaQ mutator and the RNase H genes of Escherichia coli: overlap of the promoter regions. 631 47

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

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.
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PMID:Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro. 865 73

Two basic processes are involved in protein evolution: One is amino acid replacement and another is reorganization of structural or functional units of proteins. Multidomain or multifunctional proteins are thought to have evolved by fusion of smaller structural units such as modules or domains. Reverse transcriptase (RT) is one of such fused proteins. The N-terminal part forms of globular domain with polymerase activity and the C-terminal part forms another globular domain with ribonuclease H activity (RNase H domain). There are single-domain enzymes which are homologous with the RNase H domain. The group of enzymes is called type I ribonuclease H (RNase HI). It is most likely that the ancestors of RNase HI and the polymerase domain were fused and became contemporary RT. At fusion, amino acid replacements presumably occurred at the interface of the domains to reinforce the interdomain interactions. Such replaced amino acid residues are conserved during evolution of the fused enzyme. We analyzed the pattern of amino acid replacement at each residue site in the free form, RNase HI group, and the integrated form, RNase H domain group. Then we compared the patterns between the two forms. Drastic fitting replacements of amino acid residues occurred at four of 29 residue sites involved in interdomain contact. Hydrophilic amino acid residues of the free form were substituted with hydrophobic or ambivalent ones in the integrated form. These substitutions aid in stabilizing the fused conformation by hydrophobic interactions at the interface of the domains. These observations imply that domain fusion could have occurred with only a relatively small number of adaptive amino acid substitutions.
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PMID:Adaptive amino acid replacements accompanied by domain fusion in reverse transcriptase. 907 Oct 24

The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells. The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase. Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR. Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell. Physical interactions were confirmed with purified components by means of an ELISA assay. Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected. The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM. A possible physiological significance of this type of interaction is discussed.
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PMID:HIV-1 protease inhibits its homologous reverse transcriptase by protein-protein interaction. 910 51

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.
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PMID:Human T-cell leukemia virus type 1 reverse transcriptase (RT) originates from the pro and pol open reading frames and requires the presence of RT-RNase H (RH) and RT-RH-integrase proteins for its activity. 965 93

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.
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PMID:Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination. 977 97

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.
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PMID:Hepatitis B virus: DNA polymerase activity of deletion mutants. 1020 76


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