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1994-10-08
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Document 0285
DOCN M94A0285
TI Nucleic acid binding properties of recombinant Zn2 HIV-1 nucleocapsid
protein are modulated by COOH-terminal processing.
DT 9412
AU Khan R; Giedroc DP; Department of Biochemistry and Biophysics, Texas A&M
University,; College Station 77843-2128.
SO J Biol Chem. 1994 Sep 9;269(36):22538-46. Unique Identifier : AIDSLINE
MED/94357893
AB The nucleocapsid protein (NC) of all animal retroviruses is the major
structural protein of the core ribonucleoprotein complex, bound to
genomic RNA in mature virions. In a previous report, we showed that
recombinant NC protein from HIV-1, a 71-amino-acid protein (NC71), is
apparently able to form two types of protein-nucleic acid complexes
under low [NaCl], pH 8.3 and 25 degrees C. These appeared to differ in
occluded apparent site size, napp, forming n = 8 and n = 14 complexes on
poly(A) (Dib-Hajj, F., Khan, R., and Giedroc, D. P. (1993) Protein Sci.
2, 331-243) under conditions of high and low protein-nucleotide ratios,
respectively. Here we show that both NC71-poly(A) complexes strongly
scatter light under these solution conditions. Examination of the
wavelength dependence of the light scattering at lambda < or = 320 nm
indicates that each complex is characterized by a different scattering
coefficient. Optical density measurements suggest that upon formation of
the saturated n = 8 complex, additional polynucleotide is not
incorporated into the complex over a period of hours, i.e. the n = 14
complex is not formed via redistribution of the n = 8 complex under low
salt conditions, 25 degrees C. In contrast, the n = 14 complex readily
incorporates additional protein until that sufficient to form the n = 8
complex is present. The n = 14 complex efficiently precipitates poly(A)
and shows spectral characteristics expected for an extensively
charge-neutralized nucleic acid complex. At [NC71] in excess of that
required to form the n = 8 complex, this n = 14 complex is best
described as a kinetic intermediate on the pathway to the n = 8 complex,
which forms over a period of hours under low salt conditions, 25 degrees
C. This slow kinetics of binding provides a possible explanation for the
finding that the previously observed moderate cooperativity of Zn2 NC71
binding to poly(A) (omega = 200) at pH 8.3 and 0.29 M NaCl (Khan, R.,
and Giedroc, D. P. (1992) J. Biol. Chem. 267, 6689-6695) is shown here
to represent a nonequilibrium phenomenon, apparently converting to a low
or no cooperativity complex over a period of hours. Proteolytic removal
of the COOH-terminal 14 amino acids from NC71, forming a 57-amino-acid
protein (denoted NC57), removes this apparent binding site size
heterogeneity of NC71 on poly(A). At 20 mM NaCl, NC57 binds with n = 6-7
nucleotides, in a manner which is independent of the protein-poly(A)
nucleotide ratio. The implications of these findings on processing of
the gag precursor which leads to mature NC in HIV-1 virions is
discussed.
DE Amino Acid Sequence Capsid/*METABOLISM Circular Dichroism Comparative
Study Gene Products, gag/BIOSYNTHESIS/CHEMISTRY/*METABOLISM
HIV-1/*METABOLISM Kinetics Molecular Sequence Data Poly A/*METABOLISM
Protein Binding Protein Conformation Recombinant
Proteins/BIOSYNTHESIS/CHEMISTRY/*METABOLISM RNA-Binding
Proteins/BIOSYNTHESIS/CHEMISTRY/*METABOLISM Spectrometry, Fluorescence
Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. Virion/METABOLISM
JOURNAL ARTICLE
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).