Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes.

Publication Type:

Journal Article


Nucleic Acids Res, Volume 45, Issue 15, p.9149-9163 (2017)


<p>We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6&#39;U7&#39; and 6&#39;A7&#39; on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6&#39;N7&#39; bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson-Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction.</p>

Ternary TtAgo DNA guide-DNA target complexes containing 7T8 (5XOU), 5A6 (5XQ2) and 4A5 (5XP8) bulges on the guide strand. Ternary TtAgo DNA guide-RNA target complexes containing 6’U7’ (5XPG), 6’A7’ (5XOW) and 9’U10’ (5XPA) bulges on the target strand.