Mutations at position -10 in the lambda PR promoter primarily affect conversion of the initial closed complex (RPc) to a stable, closed intermediate (RPi)

Biochemistry
M McKaneG N Gussin

Abstract

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 complex...Continue Reading

References

Mar 1, 1991·Journal of Bacteriology·H MoyleM M Susskind
Jan 1, 1985·Annual Review of Biochemistry·W R McClure
May 25, 1982·Journal of Molecular Biology·D K Hawley, W R McClure

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