Abstract
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.
Highlights
Argonaute (Ago) proteins are key players in both gene regulation and host defense
In a previous biophysical study we suggested that human Argonaute 2 uses lateral diffusion along RNA for target search[14]
Within a vast number of potential targets, Ago-guide complexes have to minimize the time spent unproductively diffusing through solution or redundantly checking off-targets, as timely regulation is crucial for both cell development and host defense[38]
Summary
Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. After binding to DNA non- from solution, the protein diffusively scans only a limited section[13,18,19,20], and dissociates into solution before rebinding to a new section. Use of such a mechanism would lead to reduced sampling redundancy, and the possibility to circumvent obstructions when proteins search for their targets
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