LacI strikes a balance between stability and inducibility.

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 Escherichia 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 2479 different Lac operators and in vivo via single-molecule experiments. We identified a mutation that enhances the specificity but reduces binding strength globally, and another mutation that makes the operator binding stronger but also reduces the specificity. However, the altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to Isopropyl β-D-1-thiogalactopyranoside (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 trade-off is between binding stability and inducibility rather than between speed and binding stability.