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

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGN
OSTI Identifier:
1430343
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nucleic Acids Research; Journal Volume: 45; Journal Issue: 15
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Sheng, Gang, Gogakos, Tasos, Wang, Jiuyu, Zhao, Hongtu, Serganov, Artem, Juranek, Stefan, Tuschl, Thomas, Patel, Dinshaw J., and Wang, Yanli. Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes. United States: N. p., 2017. Web. doi:10.1093/nar/gkx547.
Sheng, Gang, Gogakos, Tasos, Wang, Jiuyu, Zhao, Hongtu, Serganov, Artem, Juranek, Stefan, Tuschl, Thomas, Patel, Dinshaw J., & Wang, Yanli. Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes. United States. doi:10.1093/nar/gkx547.
Sheng, Gang, Gogakos, Tasos, Wang, Jiuyu, Zhao, Hongtu, Serganov, Artem, Juranek, Stefan, Tuschl, Thomas, Patel, Dinshaw J., and Wang, Yanli. Tue . "Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes". United States. doi:10.1093/nar/gkx547.
@article{osti_1430343,
title = {Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes},
author = {Sheng, Gang and Gogakos, Tasos and Wang, Jiuyu and Zhao, Hongtu and Serganov, Artem and Juranek, Stefan and Tuschl, Thomas and Patel, Dinshaw J. and Wang, Yanli},
abstractNote = {},
doi = {10.1093/nar/gkx547},
journal = {Nucleic Acids Research},
number = 15,
volume = 45,
place = {United States},
year = {Tue Jun 27 00:00:00 EDT 2017},
month = {Tue Jun 27 00:00:00 EDT 2017}
}
  • Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalyticmore » Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.« less
  • The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas twomore » critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.« less
  • Here we report on a 3.0 {angstrom} crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formationmore » of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.« less