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)

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5' of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5' end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.
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PMID:Cleavage specificities of Moloney murine leukemia virus RNase H implicated in the second strand transfer during reverse transcription. 759 16

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.
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PMID:3'-immature tRNA(Trp) is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase. 764 53

We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.
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PMID:Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain. 768 7

Region 980-1061 in human 18S rRNA was chosen on the basis of our previous results indicating, that the cross-linking sites of alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their ability to bind selectively at the desired rRNA sequences was proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Only four of the probes were able to bind to 40S subunits indicating, that the corresponding 18S rRNA sequences 980-994, 987-1001, 1025-1039 and 1032-1046 are exposed within the subunits. None of the probes inhibited tRNA-dependent binding of oligo(U) messengers to 40S subunits. Nevertheless, two probes (complementary to 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA.eIF-2.GTP was strongly affected by the same oligomers. Thus 987-1001 and 1025-1039 18S rRNA sequences are supposed to be involved in interaction with mRNA in the course of translation.
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PMID:[Functional value of 980-1061 sequences of human 18S ribosomal RNA using complementary DNA probes]. 772 52

Cassava vein mosaic virus (CVMV) was found to be widespread throughout the north-eastern region of Brazil. The complete sequence of CVMV was determined, and the genome was 8158 bp in size. A cytosolic initiator methionine tRNA (tRNA met1)-binding site that probably acts as a primer for minus-strand synthesis was present. The genome contained five open reading frames that potentially encode proteins with predicted molecular masses of 186 kDa, 9 kDa, 77 kDa, 24 kDa and 26 kDa. The putative 186 kDa protein had regions with similarity to the zinc finger-like RNA-binding domain that is a common element in the capsid proteins and similarity to the intercellular transport domain of the plant pararetroviruses. The predicted 77 kDa protein had regions with similarity to aspartic proteases, reverse transcriptase and RNase H of pararetroviruses. This gene order was confirmed by the amplification of similar PCR products from total DNA extracted from CVMV-infected cassava plants. The genomic organization of CVMV was different from the organization of either the caulimoviruses or badnaviruses. In comparisons of the regions with the reverse transcriptase motif, CVMV was grouped between the caulimoviruses and badnaviruses. It appears that CVMV is distinct from the other well-characterized plant pararetroviruses.
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PMID:Characterization of cassava vein mosaic virus: a distinct plant pararetrovirus. 773 Aug 13

We have carried out a solution study of the local conformation in a hybrid-chimeric duplex of the [sequence: see text] type (where r and D represent RNA and DNA). The object of this study was to investigate the sugar conformations at the internal junction in the hybrid-DNA octamer duplex (gccaCTGC). (GCAGTGGC)--where the lower-case letters represent RNA residues. Such duplexes represent good models for Okazaki fragments in which RNA primers are covalently extended into DNA strands during DNA replication of the lagging strand. Furthermore, this particular sequence occurs during HIV-1 retrovirus reverse transcription. The chimeric RNA-DNA strand and the complementary pure DNA strand chosen for this study result from the priming of (-)-strand DNA synthesis by tRNA(Lys) and subsequent (+)-strand DNA synthesis by reverse transcriptase prior to HIV-1 retrovirus integration. Despite the unusual specificity of the RNase H activity of reverse transcriptase, which cleaves the RNA c-a phosphodiester rather than the junction a-C linkage, we found no major structural differences among the RNA c-a phosphodiester rather than the junction a-C linkage, we found no major structural differences among the RNA sugar conformations--all RNA sugars were found in the normal C3'-endo A-form conformation. Instead, we find that the first DNA residue of the chimeric strand (5C) assumes a sugar conformation in the C4'-exo to O4'-endo range (P = 54-90 degrees). Furthermore, the hybrid segment of this duplex is more heteronomous than previously assumed for duplexes of the [sequence: see text] type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sugar conformations at hybrid duplex junctions in HIV-1 and Okazaki fragments. 838 Jul 8

The exit (E) site of the Escherichia coli ribosome was investigated using oligodeoxyribonucleotides complementary to single-stranded regions of ribosomal RNA suggested to be involved in tRNA binding in the E site [Moazed, D., & Noller, H. (1989) Cell 57, 585-597]. Radiolabeled DNA oligomers (probes) were hybridized in situ to complementary sites on the ribosomal RNA of ribosomes or ribosomal subunits, and the effects of simultaneous tRNA or antibiotic binding on probe binding were measured using a nitrocellulose filtration binding assay. Site specificity of probe binding was assured using ribonuclease H to cleave the ribosomal RNA at the site of probe binding. When 50S subunits were hybridized with a probe spanning bases 2109-2119 and deacylated tRNA was added incrementally, probe binding decreased, suggesting that the probe and tRNA competed for the same binding site or that tRNA was allosterically affecting the probe binding site. When 70S ribosomes were substituted for 50S subunits, probe binding to this site initially increased and then decreased at higher concentrations of deacylated tRNA. Titrating probe-ribosome complexes with acylated tRNA, N-acetyl-acylated tRNA, tetracycline, or chloramphenicol had no effect on probe binding. The data presented provide evidence for tRNA/rRNA interaction at or near the E site of the E. coli ribosome and suggest that a conformational change occurs in the E site when deacylated tRNA is bound to the P site. The data suggest that deacylated tRNA in the P site serves as a translocational trigger by causing the E site to change conformations, making it more available for tRNA (and probe) binding and therefore promoting translocation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for a conformational change in the exit site of the Escherichia coli ribosome upon tRNA binding. 838 94

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.
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PMID:The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site. 852 54

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).
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PMID:The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site. 859 57


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