J Virol. 1993 Jul;67(7):4037-49. Unique Identifier : AIDSLINE
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
Amino Acid Sequence Base Sequence Cloning, Molecular
HIV-1/*ENZYMOLOGY Molecular Sequence Data
Oligodeoxyribonucleotides/CHEMISTRY Ribonuclease H, Calf
Thymus/CHEMISTRY/*ISOLATION & PURIF RNA-Directed DNA
Polymerase/*CHEMISTRY RNA, Transfer, Lys/METABOLISM RNA,
Viral/METABOLISM Structure-Activity Relationship Support, Non-U.S.
Gov't Support, U.S. Gov't, P.H.S. JOURNAL ARTICLE