Argonaute bypasses cellular obstacles without hindrance during target search

Abstract

Argonaute (Ago) proteins are key players in gene regulation in eukaryotes and host defense in prokaryotes. For specific interference, Ago relies on base pairing between small nucleic acid guides and complementary target sequences. To efficiently scan nucleic acid chains for potential targets, Ago must bypass both secondary structures in mRNA and single stranded DNA as well as protein barriers. Through single-molecule FRET, we reveal that lateral diffusion is mediated mainly through protein-nucleic acid interactions, rather than interactions between the guide and targeted strand. This allows Ago to scan for targets with high efficiency but without maintaining tight contact with the DNA backbone. Real-time observations show that Ago “glides” short distances over secondary structures while using intersegmental jumps to reduce scanning redundancy and bypass protein barriers. Our single-molecule method in combination with kinetic analysis may serve as a novel platform to study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

Facilitated diffusion of Argonaute-mediated target search

“…If two targets are present on one strand, one did not only observe the addition of binding signatures with a lower FRET state, but also a shuttling signature: the transition from one binding site to the other without interruption (Figure 6(b)) [67] Since it appeared that the Ago complex did not dissociate from the strand during translocation from one target to the other target, it was suggested that lateral diffusion took place between the two targets [67]. This has also been observed for a pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo) [71,72]. Kinetic modelling suggested that if lateral diffusion would place, a linear relation would exist between distance and shuttling time.…”

“…A point worthy of note here is that the residence times for individual targets were found to be much shorter than the residence time of a single target [72]. This indicates that apparent stable binding traces contain rapid excursions away from and back to the target site.…”

“…The red line indicates a lateral diffusion fit derived from a minimal kinetic model. The data diverge from the kinetic theory beyond the blue region and follows a different trend in the green region [72]. (d) Triple target assay used to visualize whether CbAgo skips over the middle target when translocating from site A to site C. Percentages indicate the relative amount of transitions from an initial state to a final state versus the total number of transitions [72].…”

“…In cellular environments, proteins and secondary structures are likely to form road blockades, potentially blocking proteins that diffuse laterally along the strand [9]. Remarkably, Ago from the mesophile Clostridium butyricum was not found to be impeded in any way by secondary structures or protein barriers placed between the two target sites (Figure 6(i)) [72]. This suggests that sliding is not a dominant mechanism in the target search of Ago and also that the target search process itself is robust: the time to search for a target does not depend on the presence of secondary structures.…”

“…To test this out, the authors took a construct from the region where it behaved according to lateral diffusion model and a construct from the green region. The two shuttling times of the two constructs were compared at varying ionic strength [72]. If the lateral movements of Ago consisted of hopping, this would become apparent in the sensitivity to changing salt concentration.…”