Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study.
In the initiation of reverse transcription in retroviruses, nucleocapsid (NC) protein accelerates the rate of annealing of transfer RNA replication primer to a complementary sequence on the genomic RNA. In this report, we have probed the conformational changes induced by HIV-1 NC protein and domain deletion mutants in a structurally well-characterized transfer RNA, yeast tRNAPhe, as a model for the natural primer. One molar equivalent of recombinant 71 amino acid HIV-1 nucleocapsid protein (NC 1-71) is sufficient to completely inhibit the Pb2(+)-ribozyme activity of tRNAPhe at 25 degrees C, pH 7.0 and 15 mM MgCl2, Zn2 HIV-1 NC proteins which lack one or both flexible terminal domains also inhibit the ribozyme activity. 1H NMR spectra acquired for Mg(2+)-tRNAPhe suggest that NC 1-71 and NC 12-55 (lacking residues 1-11 and 56-71) inhibit the lead-ribozyme activity by only modestly altering the active site region rather than inducing large-scale unfolding of the molecule. In the absence of Mg2+, the extent of destabilization of tRNAPhe is greater but appears to be confined to internal regions of the acceptor and T psi C helices, as evidenced by the selectively enhanced exchange rates for imino protons associated with these base pairs. These findings show that NC destabilizes the folded form of tRNAPhe and by extension, other complex RNAs, in tertiary and secondary structural regions most susceptible to thermally-induced denaturation.
Khan
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Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Chang
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Kaluarachchi
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Giedroc
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D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D018360:Crystallography, X-Ray; D015683:Gene Products, gag / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D007700:Kinetics; D007854:Lead / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D019251:Nucleocapsid / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C080114:NC71 protein, Human immunodeficiency virus 1; C071884:NCP7 protein, Human immunodeficiency virus 1; D016337:RNA, Catalytic; D012346:RNA, Transfer, Amino Acyl; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D007854:Lead; D008274:Magnesium