Effects of a Single Base-pair Deletion in the Bacteriophage λ PRM Promoter : Repression of PRM by Repressor Bound at OR2 and by RNA Polymerase Bound at PR

Abstract

We have deleted a single base-pair in the -35 region of the bacteriophage λ P RM promoter. The deletion ( P RMΔ34) creates a better match of P RM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo. Since the mutation also increases the overlap between O R2 and the -35 region of P RM , binding of repressor to O R2 no longer activates, but in fact represses P RM. Finally, the mutation decreases the distance between the P RM and P R transcription start sites from 82 to 81 base-pairs. As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other. Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion. Both promoters can be occupied by RNA polymerase at the same time. Determination of K B and k f revealed that inhibition of P RM Δ34 by P R was manifest in a 100-fold decrease in the value of k f, but at the same time K B was increased tenfold. These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes. In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone. These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter. Assays of the effects of the Δ34 mutation in vivo indicate that interference between P RM and P R does not limit the rate of open complex formation at P RM in the cell. Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.