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

Abstract

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 <i>Escherichia coli</i> as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (<i>Rut</i>) sites. The map reveals many features, including promoter- and intrinsic terminator-associated <i>Rut</i> sites, bidirectional <i>Rut</i> tandems, and cofactor-dependent <i>Rut</i> sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic <i>Rut</i> 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.