Inhibition of Transcription Initiation bu IacRepressor

Initiation of transcription of the lacoperon by RNA polymerase (R) is inhibited by binding of lacrepressor (L) to an operator site which overlaps the lacpromoter (P). We have investigated repression of the lacUV5 promoter in vitrofor a choice of the repressor|mdash|operator binding constant and ranges of thermodynamic activities of L and R which appear to be relevant in vivo. Effects of [L] on the extent of formation and the kinetics of association and dissociation of abortively-initiating open complexes (RP init) were examined using fluorescence detected abortive initiation and KMnO 4chemical probing. The nitrocellulose filter assay was used to measure the dissociation rate constant and the equilibrium constant for binding for L to its operator site in the absence of R. For the chosen solution conditions, we find that both the observed velocity of abortive RNA oligomer synthesis and the KMnO 4reactivities of bases in the open region are functions of [L] and [R], demonstrating that formation of both RP initand the repressor–operator complex (PL) are reversible processes under these conditions, and requiring the use of a relaxation-to-equilibrium analysis to interpret the kinetics. The agreement between dissociation rate constants of RP initwhen challenged with either lacrepressor or heparin, and the dependences on [L] and [R] of abortive synthesis velocities at binding equilibrium and of relaxation rate constants for reversible formation of RP initfrom PL, all provide evidence for a simple competition mechanism. In this mechanism, and in contrast to recent proposals from other laboratories, lacrepressor inhibits formation of RP initand hence the observed rate of abortive product synthesis by reducing the equilibrium extent of formation of the first closed complex (RP cl), without affecting either the nature of RP initor steps in formation of RP initfrom RP cl.