Cooperative Binding of the Leucine-Responsive Regulatory Protein (Lrp) to DNA

Abstract

The leucine-responsive regulatory protein (Lrp) of Escherichia coli activates expression of a number of operons and represses expression of others. For some members of the Lrp regulon, exogenous leucine mitigates the effect of Lrp, for some it potentiates the effect of Lrp, and for others it has no effect on Lrp action. For the ilvIH operon that we study, Lrp activates expression in vivo and mediates the repression of the operon by exogenous leucine. We studied Lrp-1, a leucine-insensitive variant, to investigate mechanisms by which leucine alters Lrp action as an activator of ilvIH expression. The Asp114Glu change did not have much effect on the amount of total Lrp-1 in cells but decreased the amount of free Lrp-1 two- to threefold. Lrp monomers associate to form octamers and hexadecamers (hexadecamer form predominates at micromolar concentrations; Kd=5.27x10(-8) M), and leucine promotes the dissociation of Lrp hexadecamer to a leucine-bound octamer. By contrast, Lrp-1 exists primarily as an octamer in solution (equilibrium dissociation constant 6.5x10(-5) M) and leucine had little effect on the equilibrium. Thus, the hexadecameric form that Lrp assumes in the absence of DNA is not required for activation of the ilvIH operon. Both leucine and the lrp-1 mutation reduced the apparent affinity of Lrp binding to ilvIH DNA (contains two groups of binding sites separated by 136 bp) but they have different effects on intrinsic binding affinity and binding cooperativity. Whereas leucine reduced intrinsic binding affinities and interactions of Lrps bound at upstream and downstream regions of ilvIH DNA, it increased cooperative dimer-dimer interactions of Lrps bound to two adjacent sites. By contrast, the lrp-1 mutation did not have much effect on intrinsic binding affinities but it decreased cooperative adjacent dimer-dimer interactions and enhanced interactions of Lrps bound at upstream and downstream regions of ilvIH DNA. Our analysis is consistent with the idea that leucine enhances dimer-dimer interactions that contribute to octamer formation, concomitantly reducing dimer-dimer interactions that contribute to the longer range interactions of Lrps that are required for activation of the ilvIH promoter.