Kinetic and thermodynamic characterization of the interaction between Q beta-replicase and template RNA molecules.
The specific binding of the RNA polymerase Q beta-replicase to some of its RNA template molecules, the single-stranded RNA variant MDV and also Q beta-RNA, was studied under various conditions by using a gel-retardation assay as well as filter retention. The dissociation of the replicase-RNA complex proceeds with first-order kinetics. The dependence of the dissociation rate constant on the concentration of monovalent ions suggests that there are three contacts between the midivariant (MDV) RNA and the replicase. Through analysis of the temperature dependence of the dissociation rate constant, values of 35 and 43 kJ/mol were obtained for the activation energies of complex dissociation between Q beta-replicase and the minus (-) and plus (+) strands of MDV, respectively. The bimolecular association is of second order with high rate constants that increase when the temperature is raised and decrease at higher salt concentrations. The equilibrium constants vary between 4.10(11) M-1 and 5.10(7) M-1, according to the reaction conditions. The temperature dependence of Ka gives delta H = -39 kJ/mol for MDV- and -47 kJ/mol for MDV+. Under nearly all conditions, distinct differences in the association and dissociation rates of plus and minus strands of MDV are observed. The binding of the small variant MDV to Q beta-replicase is three orders of magnitude stronger than the binding of the natural template Q beta-RNA.
Werner
M
M
Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Federal Republic of Germany.
D003090:Coliphages / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008962:Models, Theoretical; D011777:Q beta Replicase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D013816:Thermodynamics
D046911:Macromolecular Substances; D012367:RNA, Viral; D011994:Recombinant Proteins; D011777:Q beta Replicase