Promoter search by Escherichia coli RNA polymerase on a circular DNA template.

摘要: Abstract Using the rapid-mixing/photocross-linking technique developed in our laboratory, we have investigated kinetics of interaction between Escherichia coli RNA polymerase and pAR1319, a recombinant plasmid DNA containing bacteriophage T7 A2 early promoter. By monitoring time-dependent density bound along relaxed circular molecule using this technique, been able to demonstrate kinetic evidence for linear diffusion different system from that previously described (Park, C. S., Hillel, Z., Wu, C.-W. (1982) J. Biol. Chem. 251, 6950-6956). The nonspecific association rate constant kon was measured be 7.7 x 10(4) M-1 s-1 at chain concentration 22.4 nM. taking advantage fact rapid mixing displaces protein molecules DNA, but leaves them within domain intradomain binding pAR1319 determined 8.2 s-1. Since is by radius gyration 0.22 microns, base pairs could calculated. (180 microM), estimated 4.6 In addition, mathematical model has used fit other two important constants experimental data: koff, which describes dissociation sites, D1, one-dimensional coefficient enzyme molecule. model, as ring interconnected sites together comprise "domain." polymerase, enters via three-dimensional binds each site, allowed diffuse linearly adjacent three-dimensionally on off equations occupancy site written, based general principles. solving resulting family differential equations, koff D1 were 0.3 1.5 10(-9) cm2 s-1, respectively.