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Examination Of An Equilibrium Interpretation Of Deoxyribonucleic Acid–ribonucleic Acid Hybridization Data

John O. Bishop

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1. When a constant amount of denatured DNA is annealed for a constant time with a series of different RNA concentrations, it is often observed that the reciprocal of the amount of RNA hybridized is linearly proportional to the reciprocal of the RNA concentration. This may be explained by assuming that an equilibrium is set up between free RNA and DNA on the one hand and DNA–RNA hybrid on the other. The hybridization of Escherichia coli DNA and ribosomal RNA was used to test this proposition. Rate constants were estimated from the initial rates of the forward and back reactions and compared with direct estimates of the dissociation constant. 2. The rate constants of the forward and back reactions were estimated to be 1.82mlμg-1h-1 (160lmol-1s-1) and 0.023h-1 (6.4×10-6s-1) respectively, giving a ratio k2/k1=0.013μgml-1. After 24h annealing the dissociation constant was estimated to be 0.114μgml-1, and by extrapolation to infinite time, 0.047μgml-1. 3. It is concluded that (a) equilibrium greatly favours the hybrid complex, (b) equilibrium is not established in 24h, (c) the equilibria that were directly estimated are incompatible either with the measured rates of the forward and back reactions or with the simple formulation of the reaction that was adopted, and finally (d) for these reasons the equilibrium interpretation of the linear reciprocal relationship is unsatisfactory.