An active recombinant p15 RNase H domain is functionally distinct from the RNase H domain associated with human immunodeficiency virus type 1 reverse transcriptase.
An active p15 RNase H domain, consisting of amino acids 427-560 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatography. The purified p15 RNase H domain exhibited no substrate preference for [3H]poly(rG).poly(dC) compared to [3H]poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate. Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant p15 RNase H domain was relatively nonprocessive in RNA degradative activity of the [3H]poly(rA).poly(dT) duplex. Kinetic studies using p15 RNase H showed substrate inhibition with an apparent K(i) value of 0.12 micron for the [3H]poly(rA).poly(dT) hybrid. Substrate inhibition was not observed for the HIV-1 RT-associated RNase H. The results show that the isolated p15 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H.
Evans
D B
DB
Biochemistry Research, Upjohn Laboratories, Kalamazoo, Michigan 49001.
Fan
N
N
Swaney
S M
SM
Tarpley
W G
WG
Sharma
S K
SK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H