Metal binding and activation of the ribonuclease H domain from moloney murine leukemia virus.
The RNase H family of enzymes degrades RNA in RNA.DNA hybrids in a divalent cation-dependent manner. RNases H from diverse sources such as Escherichia coli and human immunodeficiency virus (HIV) share homologous metal-binding active sites, and the activity of the RNase H domain of reverse transcriptase (RT) is required for retroviral replication. The isolated RNase H domain from HIV RT, however, is inactive. In contrast, the RNase H domain of Moloney murine leukemia virus (MMLV) is active, enabling functional studies. Unlike both E. coli RNase HI and HIV RT, the RNase H activity of MMLV RT shows greater activity in Mn(2+) than Mg(2+). We investigated the effect of mutations in five conserved active-site residues of the isolated MMLV RNase H domain. Mutations in two carboxylates eliminate metal binding while mutations in other active-site residues allow retention of metal ion affinity. Mutations that inactivate E.coli RNase HI in Mg(2+) have similar effects on the Mn(2+)-dependent activity of MMLV RNase H. These results suggest a similar one-metal catalytic mechanism for the Mn(2+)- and Mg(2+)-dependent activities of both prokaryotic and retroviral ribonucleases H.
Goedken
E R
ER
Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall, Berkeley, CA 94720, USA.
Marqusee
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D019791:Guanidine; D015497:HIV-1 / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
D002412:Cations; D008345:Manganese; D016914:Ribonuclease H; D008274:Magnesium; D019791:Guanidine