Villarruel Dujovne, M. | Search bioRxiv

bioRxiv2026

Evolution of allostery without shape shifting: Internal dynamics drives functional diversification of a transcriptional repressor superfamily

Allostery enables proteins to couple environmental signals to functional outputs, yet how allosteric mechanisms diversify during evolution remains poorly understood. Here, we address this question in the ubiquitous and functionally diverse arsenic repressor (ArsR) superfamily by integrating information-theoretic bioinformatics, structural characterization of DNA recognition and NMR measurements of fast internal dynamics. We identify conserved residues that define the structural scaffold of ArsR proteins and subfamily-specific positions that encode inducer and DNA specificity. In the persulfide sensor SqrR, the crystal structure of the DNA-bound complex reveals how operator specificity is encoded by a limited set of residues, consistent with sequence-derived predictions functionally validated by in vitro transcription assays across divergent ArsR regulators. We further show that allosteric inhibition of DNA binding in SqrR occurs without large-scale conformational rearrangements and is instead associated with changes in internal dynamics, as previously observed for the zinc sensor CzrA. Together, these results support a model in which conformational entropy preserves allosteric connectivity while relaxing sequence constraints, thereby enabling functional diversification within a protein superfamily.

biochemistry↗

bioRxiv2026

In vitro evolution of DNA operators enables multivalency protein-DNA interactions: towards programmable transcription factor regulation

In vitro transcription (IVT) systems regulated by allosteric transcription factors (aTFs) are central to emerging cell-free biosensing and synthetic biology platforms, yet their performance is often limited by suboptimal protein-DNA interactions and the need for well-characterized regulatory elements. Here, we report an in vitro evolution strategy to engineer DNA operator sequences that enables tunable aTF-DNA interactions without requiring prior detailed knowledge of the native operator or regulatory mechanism. Using a SELEX-based approach with integrated positive and counter-selection steps, we evolved non-natural operators for the sulfane sulfur-responsive transcriptional repressor SqrR. The selected sequences preserve ligand-responsive allostery, with some sequences exhibiting enhanced binding affinity and reducing transcriptional leakage. Notably, we identify operator with binding behaviors consistent with cooperative recruitment of multiple SqrR dimers, suggesting that sequence architecture can modulate aTF-DNA interactions beyond affinity alone. Incorporation of these operators into IVT circuits improves transcriptional control and dynamic range, enabling the development of ROSALIND-based sensors for sulfane sulfur species, achieving sensitive and selective detection in a fully cell-free format. More broadly, this work establishes operator evolution as a programmable strategy to optimize transcription factor-DNA interactions and expand the design space of transcription-based biosensors, including for systems lacking well-characterized genetic components.

synthetic biology↗