Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis.

Title Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis

Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation.

We have previously used site-directed mutagenesis to introduce basic residues (i.e., Arg; Lys) in the nucleic acid binding cleft of the Moloney murine leukemia virus reverse transcriptase (MMLV RT) in order to increase its template-primer (T/P) binding affinity. Three stabilizing mutations (i.e., E286R, E302K, and L435R) were identified (Yasukawa et al., 2010). Now, we studied the mechanism by ...

Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomer...

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-typ...

Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.

Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in ...