RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III.
RNase D was recently reported as a new enzymatic activity associated with HIV-1 reverse transcriptase (RT), cleaving RNA at two positions within the double-stranded region of the tRNA primer-viral RNA template complex (Ben-Artzi et al., Proc. Natl. Acad. Sci. USA 89 (1992) 927-931). This would make RNase D a fourth distinct activity of HIV-1 RT, in addition to RNA- and DNA-dependent DNA polymerase and RNase H. Using a specific substrate containing tRNA(Lys,3) hybridized to the primer binding site, we were able to detect the reported RNase D activity in our preparations of recombinant HIV-1 RT. This activity was also present in several active-site mutants of RT, suggesting that it is independent of the RNase H and polymerase functionalities of RT. Furthermore, we found that the cleavage specificity of RNase D is the same as that of RNase III isolated from E.coli. A likely explantation of these results--that the observed RNase D activity is attributable to traces of RNase III contamination--was further strengthened by the finding that the recombinant preparations of HIV-1 RT can specifically cleave a phage T7-derived double-stranded RNA processing signal, which has been used as a model substrate for detection of E.coli RNase III. Moreover, RT purified from an RNase III- strain of E.coli displayed no cleavage of the tRNA primer-RNA template complex.
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D043244:Ribonuclease III; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012330:RNA, Double-Stranded; D012357:RNA, Transfer, Lys; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli