Electron microscopy analysis of the interaction between Escherichia coli DNA-dependent RNA polymerase and the replicative form of phage fd DNA. 2. Analysis of the dissociation kinetics.
The kinetics of dissociation of the fd DNA - RNA-polymerase complex has been analyzed. Heparin was added to a solution of the enzyme - DNA complex in order to trap free polymerases. At different times after, samples were taken and analyzed by electron microscopy to determine the mean number of enzymes bound per DNA molecule. Unexpectedly, the measured dissociation is not a first-order reaction. The apparent rate constant increases with heparin concentration in the range between 0.001 and 2 mg/ml. These results strongly suggest the existence of a direct transfer process of RNA polymerase to heparin, bypassing the rate-limiting step of dissociation of the enzyme - DNA complex to free enzyme. Theoretical analysis of the direct-transfer model shows that the rate constant of dissociation should level off at high heparin concentrations: measurements of the residual transcription activity show that this is the case. From these experiments, the equilibrium constant of the DNA - RNA-polymerase complex can be determined. The value K = 10(12) M-1 which is obtained solves a striking paradox which existed because measurements performed in other laboratories indicated K = 10(14) M-1, which is greater than the equilibrium constant of the lac-repressor - lac-operator complex (=10(13) M-1).
D003090:Coliphages / Q000378:metabolism*; D004261:DNA Replication; D004278:DNA, Superhelical / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006493:Heparin; D007700:Kinetics; D008433:Mathematics; D008854:Microscopy, Electron; D014779:Virus Replication
D004278:DNA, Superhelical; D004279:DNA, Viral; D006493:Heparin; D012321:DNA-Directed RNA Polymerases