Differential effects of mutations on discrete steps in transcription initiation at the lambda PRE promoter.

The effects of cy mutations on transcription initiation at the lambda PRE promoter were determined using abortive initiation analysis (McClure, 1980). In the presence of lambda cll protein, which activates PRE, three mutations in the -10 region dramatically reduce k2, the forward rate constant for the isomerization of closed to open complexes, but only slightly affect KB, the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes. In contrast, five -35 region mutations caused decreases of 30 to 150 times in KB with much smaller effects on k2. In the absence of cll protein, the effects of mutations in the -10 region are qualitatively similar to those observed in the presence of cll protein, although the reductions in k2 are much less dramatic. In contrast, none of the mutants with defects in the -35 region is distinguishable from wildtype PRE in the absence of cll protein. Thus RNA polymerase may recognize different sequences in the -35 region in the absence of cll protein than in its presence.