A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches.
Proc Natl Acad Sci U S A
; 119(38): e2209608119, 2022 09 20.
Article
em En
| MEDLINE
| ID: mdl-36095194
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 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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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Helicases
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Técnica de Seleção de Aptâmeros
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Riboswitch
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Terminação da Transcrição Genética
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2022
Tipo de documento:
Article