title: Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities.
abstract: RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.
authors.affiliation: Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
authors.forename: S W
authors.initials: SW
authors.lastname: Blain
authors.forename: S P
authors.initials: SP
authors.lastname: Goff
mesh_terms: D015153:Blotting, Western; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D016914:Ribonuclease H