Protein · Nucleic‐Acid Reaction Kinetics

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Abstract
Methods developed to analyze experimental results concerning the binding of Escherichia coli DNA-dependent RNA polymerase [EC 2.7.7.6] to DNA at high and at low DNA concentrations, using the filter retention assay, were presented. The basic hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. At low DNA concentration: equivalence and independence of the specific binding sites; 1st-order dependence of the binding reaction on both DNA and protein concentration. At high DNA concentration: equivalence and independence of the nonspecific binding sites; no direct transfer or 1-dimensional sliding of the protein along the DNA. Comparison between theoretical predictions and experimental results at high DNA concentration will allow the determination of the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in [phage] T5 DNA). Binding experiments performed at low DNA concentration are reported: these results and the analysis which is reported allow the determination of the value of the rate constant of formation of non-filterable complexes for the system [phage] fd DNA (replicative form).cntdot.RNA-polymerase (ka = 3.3 .times. 108 M-1 s-1 in 0.1 M NaCl, 0.01 M MgCl2).