Insight into the mechanism of the stabilization of moloney murine leukaemia virus reverse transcriptase by eliminating RNase H activity.
We explored the mechanism of the stabilization of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) by eliminating RNase H activity. Without the template-primer (T/P) poly(rA)-p(dT)(15), the temperature reducing initial reverse-transcription activity by 50% over a 10-min incubation of the RNase H activity-deficient variant D524A was higher by 3.7 degrees C than that of the wild-type enzyme (WT). In the reverse transcription reaction, the K(m) values for T/P of WT and D524A were almost the same. These results suggest that elimination of RNase H activity enhanced the intrinsic thermal stability of MMLV RT rather than its affinity toward T/P.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Japan.
Masaki
M
Mizuno
Kiyoshi
K
Yasukawa
Kuniyo
K
Inouye
D004795:Enzyme Stability; D007700:Kinetics; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H