Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.
Bibliographical noteFunding Information:
We thank Ian MacRae for critical reading and Malwina Szczepaniak, Margreet Docter, Jelle van der Does, Dimitri de Roos, Anna Haagsma and Jan Wignand for technical support. C.J. was supported by Vidi (864.14.002) of the Netherlands Organization for Scientific research and an ERC Consolidator Grant (819299) of the European Research Council. M.K. and M.D. were supported by the Netherlands Organization for Scientific Research, as part of the Frontiers in Nanoscience program. M.D. acknowledges financial support from a TU Delft startup grant. J.v.d.O. was financially supported by two grants from the Netherlands Organization of Scientific Research (NWO; ECHO grant 711.013.002 and NWO-TOP grant 714.015.001).
© 2019, The Author(s).