A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.

Automated summary

In this study, a screening scheme called Helicase-SELEX was developed to explore the requirements of helicases for substrates and cofactors on a global scale. Using the transcription termination helicase Rho of E.coli as a prototype, researchers generated a genome-wide map of Rho utilization sites and developed synthetic Rut sites. This study illustrates the potential and flexibility of Helicase-SELEX in fundamental and synthetic biology discovery.