EQUILIBRIUM AND KINETIC-PARAMETERS OF THE SEQUENCE-SPECIFIC INTERACTION OF ESCHERICHIA-COLI RNA-POLYMERASE WITH NONTEMPLATE STRAND OLIGODEOXYRIBONUCLEOTIDES

The specific recognition by Escherichia coli RNA polymerase of single- stranded oligodeoxyribonucleotides (oligos) with the sequence of the - 10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopur ine residues incorporated in the oligos. The effects of salt on the ra tes of formation and dissociation of RNA polymerase oligo complexes ar e relatively small, from which we conclude that electrostatic interact ions contribute minimally to the favorable binding free energy. From t he convex temperature dependence of In K-a (K-a is the equilibrium ass ociation constant), we infer that a large apparent negative heat capac ity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is inte rpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is mor e than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change i n the RNA polymerase. Oligo binding was shown to compete with the inte raction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase.