We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg 2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavagecompetent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.bacterial Argonaute | catalytic mechanism | DNA guide-DNA target A rgonaute (Ago) proteins, critical components of the RNAinduced silencing complex, play a key role in guide strandmediated target RNA recognition, cleavage, and product release (reviewed in refs. 1-3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4-7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme's Asp-Asp-Asp/His catalytic triad (12-15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16-18). Such Mg 2+ cation-mediated endonucleolytic cleavage of the target RNA strand (19,20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson-Crick pairing of the guide and target strands spanning the seed segment (positions 2-2′ to 8-8′) and the cleavage site (10′-11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8,9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagati...

The yeast UPF1, UPF2 and UPF3 genes encode transacting factors of the nonsense-mediated mRNA decay pathway. In addition, the upf1D strain demonstrates a nonsense suppression phenotype and Upf1p has been shown to interact with the release factors eRF1 and eRF3. In this report, we show that both upf2D and upf3D strains demonstrate a nonsense suppression phenotype independent of their effect on mRNA turnover. We also demonstrate that Upf2p and Upf3p interact with eRF3, and that their ability to bind eRF3 correlates with their ability to complement the nonsense suppression phenotype. In vitro experiments demonstrate that Upf2p, Upf3p and eRF1 compete with each other for interacting with eRF3. Conversely, Upf1p binds to a different region of eRF3 and can form a complex with these factors. These results suggest a sequential surveillance complex assembly pathway, which occurs during the premature translation termination process. We propose that the observed nonsense suppression phenotype in the upfD strains can be attributed to a defect in the surveillance complex assembly.

No abstract