Kinetic studies of the modulation of ada promoter activity by upstream elements

We have determined the kinetics of initiation of transcription of the wild-type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M-1 and 1.7 x 10(-2) s-1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild-type sequence with an up-mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (-0.05), leading to a 12-fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)