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

---

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

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.
...
PMID:The isolated RNase H domain of murine leukemia virus reverse transcriptase. Retention of activity with concomitant loss of specificity. 926 41

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.
...
PMID:Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization. 944 85

We have cloned, expressed, and purified to electrophoretic homogeneity a human RNase H. The enzyme has a molecular weight of 32 kDa, is Mg2+ dependent, and is inhibited by Mn2+ and N-ethylmaleimide. Its molecular weight and cleavage characteristics are consistent with type 2 human RNase H. The human RNase H we have cloned is highly homologous to Escherichia coli RNase HI (33.6% amino acid identity) and to other RNase H enzymes homologous to E. coli RNase HI. The enzyme is encoded by a single gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues.
...
PMID:Molecular cloning and expression of cDNA for human RNase H. 951 96

Retroviral reverse transcriptase-associated RNase H enzymes are responsible for degradation of viral RNA, including removal of the tRNA primer after plus-strand strong-stop synthesis and cleavage of the polypurine tract primer. These activities are required for the complex viral replication and result in generation of the long terminal repeats. The human immunodeficiency virus type 1 (HIV-1) RNase H domain has been expressed independently of the polymerase domain and possesses Mn2+-dependent activity with a hexahistidine tag. The isolated domain maintains the ability to specifically remove a tRNA primer mimic. In this study, the substrate determinants for recognition of the cognate tRNA3Lys are defined. Model substrates were constructed which mimic the RNA-DNA hybrid obtained from plus-strand strong-stop synthesis. Deletion substrates containing only 12, 9, or 6 positions of the tRNA primer were capable of being cleaved by the isolated RNase H domain. Mismatch and bromodeoxyuridine mutagenesis analysis indicated that positions 2, 3, 4, and 6, when mutated, affected the specificity of RNase H activity. Substitution substrates indicated that positions 4 and 6 within the RNA primer were important for recognition and cleavage by the HIV-1 isolated RNase H domain. Moloney murine leukemia virus-HIV-1 hybrid substrates were constructed which demonstrated that changes to HIV-1 sequences at positions 4 and 6 were sufficient but not optimal for regaining cleavage by the isolated HIV-1 RNase H domain. Optimal site-specific cleavage between the terminal ribonucleotide A and ribonucleotide C requires additional sequences beyond the first six positions but less than nine.
...
PMID:Sequence requirements for removal of tRNA by an isolated human immunodeficiency virus type 1 RNase H domain. 965 29

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
...
PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

Ribonucleases H (RNases H) comprise a family of metal-dependent enzymes that catalyze the hydrolysis of the 3'-O---P bond of RNA in RNA.DNA hybrids. The mechanism by which RNases H use active-site metal(s) for catalysis is unclear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HIV RNase H domain, two models explaining RNase H metal dependence have been proposed: a one-metal mechanism and a two-metal mechanism. In this paper, we show that the Mn2+-dependent activity of E. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the presence of Mn2+ is complex, with activation and inhibition of the enzyme at low and high Mn2+ concentrations, respectively. Mutations at Asp-134 result in a partial loss of this inhibition, with little effect on activation. Neutralization of His-124 by mutation to Ala results in an enzyme with a significantly decreased specific activity and an absolute loss of Mn2+ inhibition. Inhibition by high Mn2+ concentrations is shown to be due to a reduction in kcat; this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" model explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory.
...
PMID:Activation/attenuation model for RNase H. A one-metal mechanism with second-metal inhibition. 985 71

Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.
...
PMID:Identification of the genes encoding Mn2+-dependent RNase HII and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families. 988

We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.
...
PMID:Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria. 1036 2

Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3Lys.
...
PMID:RNase H requirements for the second strand transfer reaction of human immunodeficiency virus type 1 reverse transcription. 1040 Jul 54

Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates. Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed. Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT. Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results. However, only one Mg2+ ion is bound in the RNase H catalytic center. Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain. The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported.
...
PMID:Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry. 1076 38


<< Previous 1 2 3 4 5 6 7 Next >>

---