Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial reverse transcriptase is responsible for the synthesis of multicopy single-stranded DNA (msDNA). Reverse transcriptases from retron-Ec73 and retron-Ec107 do not contain an RNase H domain. Cellular RNase H is therefore considered to be required to make the mature form of msDNA. We found that RNase HI, but not RNase HII, is required for the production of the mature form of both msDNAs.
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PMID:The role of ribonuclease H in multicopy single-stranded DNA synthesis in retron-Ec73 and retron-Ec107 of Escherichia coli. 752 2

Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain.
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PMID:Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity. 753 29

A single RNase H enzyme was detected in extracts of Streptococcus pneumoniae. The gene encoding this enzyme was cloned and expressed in Escherichia coli, as demonstrated by its ability to complement a double-mutant rnhA recC strain. Sequence analysis of the cloned DNA revealed an open reading frame of 290 codons that encodes a polypeptide of 31.9 kDa. The predicted protein exhibits a low level of homology (19% identity of amino acid residues) to RNase HII encoded by rnhB of E. coli. Identification of the S. pneumoniae RNase HII translation start site by amino-terminal sequencing of the protein and of mRNA start sites by primer extension with reverse transcriptase showed that the major transcript encoding rnhB begins at the protein start site. Comparison of the S. pneumoniae and E. coli RNase HII sequences and sequences of other, putative bacterial rnhB gene products surmised from sequencing data revealed three conserved motifs. Use of these motifs to search for homologous genes in eucaryotes demonstrated the presence of rnhB genes in a yeast and a roundworm. Partial rnhB gene sequences were detected among expressed sequences of mouse and human cells. From these data, it appears that RNase HII is universally present in living cells.
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PMID:The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes. 919 Jul 96

We have cloned and functionally characterized the RNase H1 gene from D. melanogaster. The longest open reading frame consists of 5 exons that encode a 333 amino acid protein with a molecular mass of 37.1 kDa. This is the first demonstration of specific nuclease activity of a cloned RNase gene from a multicellular higher eukaryote. No additional proteins or cofactors are required for this nuclease activity. Comparison of Drosophila RNase H1 amino acid sequence to that of other cellular eukaryotic homologs reveals the presence of three evolutionarily distinct domains. The N- and C-terminal conserved domains are connected by a highly variable domain. The C-terminal domain has high amino acid similarity to bacterial RNase HI and the RNase H domain of retroviral reverse transcriptase, while the N-terminus, of unknown function, is similar to the P6 translational activator of caulimoviruses.
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PMID:Functional characterization of RNase H1 from Drosophila melanogaster. 939 56

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.
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PMID:Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT. 1616 98

We report here crystal structures of human RNase H1 complexed with an RNA/DNA substrate. Unlike B. halodurans RNase H1, human RNase H1 has a basic protrusion, which forms a DNA-binding channel and together with the conserved phosphate-binding pocket confers specificity for the B form and 2'-deoxy DNA. The RNA strand is recognized by four consecutive 2'-OH groups and cleaved by a two-metal ion mechanism. Although RNase H1 is overall positively charged, the substrate interface is neutral to acidic in character, which likely contributes to the catalytic specificity. Positions of the scissile phosphate and two catalytic metal ions are interdependent and highly coupled. Modeling of HIV reverse transcriptase (RT) with RNA/DNA in its RNase H active site suggests that the substrate cannot simultaneously occupy the polymerase active site and must undergo a conformational change to toggle between the two catalytic centers. The region that accommodates this conformational change offers a target to develop HIV-specific inhibitors.
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PMID:Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. 1796 65

Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg(2+) or Mn(2+) ions. The isolated RNase H domain of HIV-1 RT (RNH(HIV)) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNH(HIV) is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNH(HIV) and BstNTD-RNH(HIV), were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNH(HIV) and exhibited enzymatic activity in the presence of Mn(2+) ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg(2+) ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn(2+)-dependent activity of RNH(HIV) but do not restore the Mg(2+)-dependent activity of RNH(HIV).
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PMID:Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2. 2567 83