Equilibrium and stopped-flow kinetic studies of interaction between T7 RNA polymerase and its promoters measured by protein and 2-aminopurine fluorescence changes

The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods, To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes, The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (-4 to +4), measured structural changes in the promoter DNA, such as DNA melting, The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism, E + D reversible arrow(K)1 ED(a) (k)-2 reversible arrow(k)2 ED(b) where the RNA polymerase forms an initial weak ED, complex rapidly with an equilibrium association constant K-1, The ED(a) complex then undergoes a conformational change to ED(1), wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step, The isomerization of ED(a) to ED(b) is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation, To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, Phi 10 and Phi 3.8, were conducted, The results indicate that kinetics, the bimolecular rate constant of DNA binding, k(on) (K1K2), and the dissociation rate constant, k(off) (h(-2)), and thermodynamics, the equilibrium constants of the two steps (K-1 and k(2)/k(-2)) both play a role in modulating the transcriptional efficiency at the level of DNA binding, Thus, the 2-fold lower k(on), the 4-fold higher k(off), and the 2-5-fold weaker equilibrium interactions together make Phi 3.8 a weaker promoter relative to Phi 10.