Sequence requirements for removal of tRNA by an isolated human immunodeficiency virus type 1 RNase H domain.
Retroviral reverse transcriptase-associated RNase H enzymes are responsible for degradation of viral RNA, including removal of the tRNA primer after plus-strand strong-stop synthesis and cleavage of the polypurine tract primer. These activities are required for the complex viral replication and result in generation of the long terminal repeats. The human immunodeficiency virus type 1 (HIV-1) RNase H domain has been expressed independently of the polymerase domain and possesses Mn2+-dependent activity with a hexahistidine tag. The isolated domain maintains the ability to specifically remove a tRNA primer mimic. In this study, the substrate determinants for recognition of the cognate tRNA3Lys are defined. Model substrates were constructed which mimic the RNA-DNA hybrid obtained from plus-strand strong-stop synthesis. Deletion substrates containing only 12, 9, or 6 positions of the tRNA primer were capable of being cleaved by the isolated RNase H domain. Mismatch and bromodeoxyuridine mutagenesis analysis indicated that positions 2, 3, 4, and 6, when mutated, affected the specificity of RNase H activity. Substitution substrates indicated that positions 4 and 6 within the RNA primer were important for recognition and cleavage by the HIV-1 isolated RNase H domain. Moloney murine leukemia virus-HIV-1 hybrid substrates were constructed which demonstrated that changes to HIV-1 sequences at positions 4 and 6 were sufficient but not optimal for regaining cleavage by the isolated HIV-1 RNase H domain. Optimal site-specific cleavage between the terminal ribonucleotide A and ribonucleotide C requires additional sequences beyond the first six positions but less than nine.
Smith
C M
CM
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Leon
O
O
Smith
J S
JS
Roth
M J
MJ
D001665:Binding Sites; D001973:Bromodeoxyuridine; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009052:Leukemia Virus, Murine; D016296:Mutagenesis; D012313:RNA; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D012361:RNA, Transfer, Pro / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C034477:RNA primers; D012346:RNA, Transfer, Amino Acyl; D012361:RNA, Transfer, Pro; D012313:RNA; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D001973:Bromodeoxyuridine