Heteronuclear NMR studies of the interaction of tRNA(3)(Lys) with HIV-1 nucleocapsid protein

Reverse transcription of HIV-1 viral RNA uses human tRNA(3)(Lys) as a prime r. Recombinant tRNA(3)(Lys) was previously overexpressed in Escherichia col i, N-15-labelled and purified for NMR studies. It was shown to be functiona l for priming of HIV-1 reverse transcription. Using heteronuclear 2D and 3D NMR, we have been able to assign almost all the imino groups in the helica l regions and involved in the tertiary base interactions of tRNA(3)(Lys). T his crucial step enabled us to address the question of the annealing mechan ism of tRNA(3)(Lys) by the nucleocapsid protein (NC) using heteronuclear NM R. Moreover, structural aspects of the tRNA(3)(Lys)/ (12-53)NCp7 interactio n have been characterised. The (12-53)NCp7 protein binds preferentially to the inside of the L-shape of the tRNA(3)(Lys), on the acceptor and D stems, and at the level of the tertiary interactions between the D and T-Psi -C l oops. (12-53)NCp7 binding does not induce the melting of any single base-pa ir or unwinding of the tRNA(3)(Lys) helical domains. Moreover, NMR provides a unique means to investigate the base-pairs that are destabilised by (12- 53)NCp7 binding. Indeed, the measurements of the longitudinal relaxation ti me T-1 and of the exchange time of the imino protons revealed two major reg ions sensitive to catalysis by the protein, namely the G6-U67 and T54(A58) pairs. It is interesting that for the biological role of the NC protein, th ese pairs could be the starting points of the tRNA melting required for the hybridisation to the viral RNA. (C) 2001 Academic Press.