NMR studies of the allosteric effectors of the lac operon

Abstract

The aim of this thesis is to characterize the regulatory mechanism of the Lac repressor which is the molecular switch of the lac operon. Lac repressor binds to its cognate DNA operator and inhibits transcription. When an inducer binds to the protein, it triggers a conformational change that releases the protein from the operator. Thus the DNA operator and inducer are two allosteric effectors of the Lac repressor. In the first part of this work the structures of a dimeric mutant of the Lac repressor DNA binding domain (‘headpiece’) complexed with the auxiliary operators O2 and O3 were determined by NMR spectroscopy. To understand the mechanism of repressor-operator recognition these structures were compared to the previously determined structures of headpiece bound to the main operator O1 and to non-specific DNA. Headpiece shows significant conformational plasticity to compensate for small changes in its cognate DNA sequences. The difference in the affinities of the Lac repressor for its natural operators can be well explained by the current structures. To gain insight into the allosteric link between inducer binding and DNA affinity, deuterated dimeric Lac repressor and isolated ‘core’ domain, containing only the inducer binding site, were produced. NMR chemical shift changes between various regulatory states of the intact Lac repressor dimer (free, protein-inducer complex, protein-DNA complex, and a ternary protein-DNA-inducer complex) could be monitored. When mapped on available X-ray structures, they show the allosteric connection between inducer binding and DNA affinity and the residues involved in this signaling path. Our findings show that the dimeric Lac repressor is in a dynamic equilibrium between two conformational states, which can be switched to either the operator bound and the inducer bound states by the addition of either inducer. This picture is gives strong molecular support for the classical model of allostery of the Lac repressor proposed by J. Monod, J. Wyman and J-P. Changeux. Furthermore data from ternary complex, revealed how inducer binding results in the disruption of the contacts between the inducer binding domain and DNA binding domains, thereby destabilizing the dimerization interface within the DNA binding domain. This causes the release of the Lac repressor from the operator DNA.