Heteronuclear NMR studies of the interaction of tRNA(Lys)3 with HIV-1 nucleocapsid protein.
Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Recombinant tRNA(Lys)3 was previously overexpressed in Escherichia coli, 15N-labelled and purified for NMR studies. It was shown to be functional 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 helical regions and involved in the tertiary base interactions of tRNA(Lys)3. This crucial step enabled us to address the question of the annealing mechanism of tRNA(Lys)3 by the nucleocapsid protein (NC) using heteronuclear NMR. Moreover, structural aspects of the tRNA(Lys)3/(12-53)NCp7 interaction have been characterised. The (12-53)NCp7 protein binds preferentially to the inside of the L-shape of the tRNA(Lys)3, on the acceptor and D stems, and at the level of the tertiary interactions between the D and T-psi-C loops. (12-53)NCp7 binding does not induce the melting of any single base-pair or unwinding of the tRNA(Lys)3 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 time T1 and of the exchange time of the imino protons revealed two major regions 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, these pairs could be the starting points of the tRNA melting required for the hybridisation to the viral RNA.
Tisné
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Laboratoire de Cristallographie et RMN Biologiques, EP 2075 CNRS Faculté de Pharmacie, 4 avenue de l'Observatoire, Paris, 75006, France. tisne@pharmacie.univ-paris5.fr
Roques
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BP
Dardel
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D000926:Anticodon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011522:Protons; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D000926:Anticodon; D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D010446:Peptide Fragments; D011522:Protons; D012357:RNA, Transfer, Lys; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D009584:Nitrogen