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)

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.
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PMID:Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition. 14 Jan 66

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

Intact primer RNA for discontinuous DNA replication of Escherichia coli has been detected by specific labeling in vitro of its 5'-terminal tri- (or di-) phosphate group with vaccinia guanylyltransferase and [alpha-32P]GTP. A mutant defective either in RNase H or in both RNase H and DNA polymerase I accumulated about 10 or 30 times more intact primer RNA, respectively, than wild-type cells. The primers started with purine in an A to G ratio of 5 and the most abundant 5'-terminal dinucleotide sequence was (p)ppA-Pu. The chain length of the intact primer RNA was approximately 10 to 12 nucleotide residues. The structural properties of the E. coli primer RNa resemble those of the eukaryotic primer RNA.
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PMID:Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine. 241 35

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.
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PMID:Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. 283 68

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

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the 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 abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 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 same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.
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PMID:Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes. 906 Oct 30

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.
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PMID:De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. 1062 48

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
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PMID:Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities. 1081 27

Cell-free extracts prepared from S. cerevisiae cells were incubated in the presence of [alpha-32P]-labeled ATP, CTP, GTP or UTP. An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3' end in the presence of ATP or UTP but not with CTP or GTP. This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this approximately 30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA. These two lines of evidence show that this approximately 30S RNA is not structurally related to ribosomal RNA gene repeat. The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells. These data show that the enzyme responsible for adenylation of this approximately 30S RNA is distinct from mRNA poly(A) polymerase. While the human SRP RNA 3' adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme.
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PMID:Identification of a approximately 30S size non-ribosomal Saccharomyces cerevisiae RNA that is rapidly labeled on its 3' end by ATP or UTP. 1125 4