LacI locates its operator sequence by combining three-dimensional diffusion through the cytoplasm with one-dimensional sliding along DNA. Earlier work has suggested that transcription factors may solve the speed-stability paradox by switching between two conformations: a search state, optimized for rapid scanning of DNA, and a recognition state, optimized for strong and specific binding to target sequences. The flexible “hinge” region of LacI is thought to play a key role in the conformational switch. In this study, we shift the balance between these conformations to study their effect on DNA-binding specificity and target-search kinetics.

Using molecular dynamics simulations, we designed two LacI mutants predicted to favor different conformational states. One mutation (V52A) was expected to stabilize the hinge helix and promote the recognition conformation, while another (Q55N) was predicted to favor the search conformation by destabilizing the hinge. We characterized the mutants using a combination of large-scale protein-binding microarrays and single-molecule experiments in living cells.

The protein-binding microarrays allowed us to measure association and dissociation kinetics for LacI binding to thousands of operator variants simultaneously. The Q55N mutant increased sequence specificity but weakened overall binding strength, whereas the V52A mutant strengthened operator binding but reduced specificity. To determine how these changes affected LacI inside living cells, we tracked single molecules in E. coli. Based on classical models, one might expect the more strongly binding V52A mutant to spend more time trapped on nonspecific DNA and therefore search more slowly. However, the experimental effect on search kinetics was modest, suggesting that the search time is a rather stable parameter.

The strongest phenotype was observed during IPTG induction. LacI normally releases its operator when IPTG is present, thereby allowing expression of the lac operon. The V52A mutant, which binds more strongly, became harder to induce and remained bound for longer than wild-type LacI.

Together, the results suggest that the evolutionary compromise faced by LacI is not primarily between search speed and binding stability, as often assumed. Rather, our data support a trade-off between binding stability and inducibility. A transcription factor that binds too tightly may still find its targets efficiently, but it becomes less responsive to regulatory signals that should trigger its release.

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