A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322.

In order to follow the kinetics of the initiation of transcription by the E. coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7). In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model. This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3). The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.). The activation energy of isomerization is 13 Kcal/mole. Both k2 and k-2 are increasing with pH. The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.