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Application of band centrifugation to the study of the assembly of alfalfa mosaic virus
Author(s) -
Driedonks René A.,
Joe Michael K. K. Tjok,
Mellema Jan E.
Publication year - 1980
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.1980.360190310
Subject(s) - chemistry , centrifugation , diffusion , elongation , rna , alfalfa mosaic virus , particle (ecology) , crystallography , analytical chemistry (journal) , coat protein , thermodynamics , chromatography , biochemistry , physics , materials science , oceanography , gene , metallurgy , ultimate tensile strength , geology
A band‐centrifugation method for the analysis of an assembly reaction of a simple virus from its RNA and protein is described. The experiment was carried out by sedimenting a band of viral RNA through a solution of depolymerized coat protein. The resulting radial distribution of the reaction products, followed as a function of time, was analyzed by a computer simulation of the series of reaction. This method is based on a numerical solution of the continuity equation for the sedimentation–diffusion process [Claverie, J.‐M., Dreux, H. & Cohen, R. (1975) Biopolymers 14 , 1685–1700; Cohen, R. & Claverie, J.‐M. (1975) Biopolymers 14 , 1701–1716]. A numerical method for the simulation of the chemical reaction is derived. From the simulated reaction series, equilibrium constants emerge for the successive addition of protein subunits to the growing nucleoprotein particle. The method is applied to the assembly of alfalfa mosaic virus. If the reaction between RNA and protein is carried out in 0.32 M CsCl, pH 7.0, two stages during particle growth are resolved, each characterized by an equilibrium constant K . The determined values for K range from 5 × 10 5 to 3 × 10 6 l. mol −1 . The existence of these two stages may have a structural implication in the assembly, as they likely represent an elongation and a termination stage. If the reaction is carried out under more favorable conditions (0.25 M CsCl, pH 7.0), a kinetic constant of at least 10 5 l. mol −1 sec −1 is derived for each reaction step. Under these conditions the assembly appears to be completed within 1 min, which is too fast to detect distinct stages by band sedimentation.