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 K(B) and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 107 M-1 and 1.7 x 10-2 s-1, respectively, at 37°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 K(B)·k2 product) remains almost constant, because the maximum value of K(B) 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 K(B) 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 K(B) 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. By two-dimensional polyacrylamide gel electrophoresis of the ligation products of two 31 bp oligonculeotides harboring the wild-type and the mutant sequences, we have shown that the mutation induces an increased bending or flexibility of the 31mer. We suggest that this increased flexibility facilitates the wrapping of the DNA around the RNA polymerase in the initiation complex, thus leading to an increased efficiency of the initiation of transcription observed for the mutant promoter.