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 DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.
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PMID:Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. 170 16

Structural and thermodynamic aspects of alkaline earth metal dication (Mg(2+), Ca(2+), Sr(2+), Ba(2+)) binding to E. coli ribonuclease H1 (RNase H1) have been investigated using both experimental and theoretical methods. The various metal-binding modes of the enzyme were explored using classical molecular dynamics simulations, and relative binding free energies were subsequently evaluated by free energy simulations. The trends in the free energies of model systems based on the simulation structures were subsequently verified using a combination of density functional theory and continuum dielectric methods. The calculations provide a physical basis for the experimental results and suggest plausible role(s) for the metal cation and the catalytically important acidic residues in protein function. Magnesium ion indirectly activates water attack of the phosphorus atom by freeing one of the active site carboxylate residues, D70, to act as a general base through its four first-shell water molecules, which prevent D70 from binding directly to Mg(2+). Calcium ion, on the other hand, inhibits enzyme activity by preventing D70 from acting as a general base through bidentate interactions with both carboxylate oxygen atoms of D70. These additional interactions to D70, in addition to the D10 and E48 monodentate interactions found for Mg(2+), enable Ca(2+) to bind tighter than the other divalent ions. However, a bare Mg(2+) ion with two or less water molecules in the first shell could bind directly to the three active-site carboxylates, in particular D70, thus inhibiting enzymatic activity. The present analyses and results could be generalized to other members of the RNase H family that possess the same structural fold and show similar metal-binding site and Mg(2+)-dependent activity.
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PMID:A combined experimental and theoretical study of divalent metal ion selectivity and function in proteins: application to E. coli ribonuclease H1. 1288 61

Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.
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PMID:The parathyroid hormone mRNA 3'-untranslated region AU-rich element is an unstructured functional element. 1458 48

Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.
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PMID:Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains. 1753 63

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism.
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PMID:Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism. 2810 Jun 93