Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 119, Issue 38, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2209608119
Keywords
helicases; riboswitches; transcription termination; Rho
Categories
Funding
- France Genomique [ANR-10-INBS09]
- Agence Nationale de la Recherche (ANR) [ANR-15-CE11-0024-2, ANR-19-CE44-0009-01]
- Agence Nationale de la Recherche (ANR) [ANR-15-CE11-0024] Funding Source: Agence Nationale de la Recherche (ANR)
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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.
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.
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