Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain.
We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.
Smith
J S
JS
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
Roth
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H