title: A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active.
abstract: We report here a human immunodeficiency virus type 1 (HIV-1) recombinant ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its isolation by affinity chromatography. The purified protein is active in hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of recent reports of similar HIV-1 RNase H domains which were enzymatically inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R., Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80; Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that a stretch of 20-30 residues immediately upstream of the polymerase-RNase H junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for productive binding and alignment of the hybrid RNA-DNA substrate. The active HIV-1 RNase H domain is suitable for structural analysis, thereby providing a unique active molecule to better understand the structural basis for the functional organization of RNase associated with the HIV-1 reverse transcriptase.
authors.affiliation: Department of Biochemistry, Upjohn Laboratories, Kalamazoo, Michigan 49001.
authors.forename: D B
authors.initials: DB
authors.lastname: Evans
authors.forename: K
authors.initials: K
authors.lastname: Brawn
authors.forename: M R
authors.initials: MR
authors.lastname: Deibel
authors.forename: W G
authors.initials: WG
authors.lastname: Tarpley
authors.forename: S K
authors.initials: SK
authors.lastname: Sharma
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H