| Abstract: |
We report on crystal structures of ternary Thermus thermophilusArgonaute (TtAgo) complexes with 5'-phosphorylated guide DNAand a series of DNA targets. These ternary complex structures ofcleavage-incompatible, cleavage-compatible, and postcleavagestates solved at improved resolution up to 2.2 A have providedmolecular insights into the orchestrated positioning of catalyticresidues, a pair of Mg2+ cations, and the putative water nucleo-phile positioned for in-line attack on the cleavable phosphate forTtAgo-mediated target cleavage by a RNase H-type mechanism. Inaddition, these ternary complex structures have provided insightsinto protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatiblestates, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage,the seed segment forms a stable duplex with the complementarysegment of the target strand. |
