title: Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.
abstract: Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Keck
authors.forename: S
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology*; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H