The lac repressor strikes a balance between inducibility and strength of repression

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2,479 different Lac operators and in vivo via single-molecule experiments. We conclude that a hinge region mutation causing less helix propensity enhances the specificity but reduces binding strength globally, while a hinge region mutation causing higher helix propensity has opposite effects. However, altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity-inducibility trade-offs in LacI, our data support the model in which the hinge region governs a trade-off between binding stability and inducibility rather than between speed and binding stability.