Characterization of Moloney murine leukaemia virus/avian myeloblastosis virus chimeric reverse transcriptases.
Reverse transcriptases (RTs) from Moloney murine leukaemia virus (MMLV) and avian myeloblastosis virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.
Yasukawa
Kiyoshi
K
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. yasukawa@kais.kyoto-u.ac.jp
Mizuno
Masaki
M
Inouye
Kuniyo
K
D000595:Amino Acid Sequence; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H