Nucleic Acids Research, Vol 24, Issue 18 3568-3575, Copyright © 1996 by Oxford University Press
R Khan, HO Chang, K Kaluarachchi and DP Giedroc
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.
ARTICLES
Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
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