Mechanisms of the Binding/Dissociation Acceleration of the Target–Guide Interaction by Thermus thermophilus Argonaute

Abstract

Prokaryotic Argonaute facilitates the target recognition process by the guide strand via a still unknown mechanism. Using single‐molecule fluorescence resonance energy transfer and systematic mutagenesis of Thermus thermophilus Argonaute and guide‐target base pairing, we study the kinetic roles of various structural features of guide strand in the prokaryotic Argonaute. We reveal that the 5′‐end anchoring of the guide strand, and the sequence complementarity in the seed‐ and mid‐regions greatly accelerate the target binding, and that this acceleration is due to the reduction of the entropic barrier of the binding process. Our data further suggest that the dynamic anchoring of the guide strand to the PAZ domain, which is coupled to target dissociation, does not accompany the partial unwinding of the guide‐target duplex.