Mutations at Position −10 in the λ PR Promoter Primarily Affect Conversion of the Initial Closed Complex (RPc) to a Stable, Closed Intermediate (RPi)

The effects of mutations of --10 T:A to A:T, C:G, or G:C in the lambda P(R) promoter on formation of transcriptionally competent open complexes were studied by DNAse I footprinting, KMnO(4)-sensitivity, and abortive initiation kinetic analysis. The mutations --10A (T:A --> A:T) and --10C significantly reduce k(f), the composite rate constant for conversion of closed complexes (RP(c)) to open complexes (RP(o)) but do not affect K(B), the equilibrium constant for formation of closed complexes. Unlike the other mutants or wild-type P(R), the mutation with the largest effect on open complex formation, --10G (T:A --> G:C), substantially decreases the occupancy of the promoter. When reduced occupancy is taken into account, the calculated effect of the mutation on k(f) is a 20-fold reduction. Analysis of open complex formation by a three-step pathway that includes an additional intermediate, RP(i), indicates that the primary effect of all three mutations is a reduction in the rate of isomerization of RP(c) to RP(i), which precedes DNA strand separation. Thus, RNA polymerase holoenzyme must recognize specific base pairs in the --10 region of P(R) while the DNA is still double-stranded. Comparison of the observed level of stable complexes (RP(i) plus RP(o)) with the level of productive complexes (RP(o)) indicates that the --10G mutation may also affect the equilibrium between RP(i) and RP(o) at 37 degrees. Open complexes formed at the three mutant promoters are approximately 3-5 times less stable at 37 degrees than those formed at wild-type P(R).