PDBsum entry 3hm9

Nucleic acid binding protein/DNA/RNA

PDB id

3hm9

Contents

			Protein chain

					654 a.a.

			DNA/RNA

			Metals

					_MG ×2

References listed in PDB file

Key reference

Title

Nucleation, Propagation and cleavage of target rnas in ago silencing complexes.

Authors

Y.Wang, S.Juranek, H.Li, G.Sheng, G.S.Wardle, T.Tuschl, D.J.Patel.

Ref.

Nature, 2009, 461, 754-761. [DOI no: 10.1038/nature08434]

PubMed id

19812667

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

Abstract

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.



	Figure 4. Figure 4: Effect of complementarity and length on target DNA cleavage by T. thermophilus Ago. Cleavage reactions were performed as described in the Methods, and products were resolved on denaturing polyacrylamide gels; for DNA sequences, see Supplementary Table 4. a, Schematic of the reference DNA duplex utilized for length variation experiments; the cleavage site is indicated by an arrow, the position of the ^32P label by an asterisk. b, Shortening of the target DNA from its 5' end. Alterations of the target DNA and corresponding paired structure are illustrated to the left. Target DNA cleavage was performed at 65 °C rather than 75 °C to facilitate hybridization of shortened targets. nt, nucleotides. c, Positional variation of 15-nucleotide target DNAs. For labelling and reaction conditions, see b.		Figure 5. Figure 5: Effect of sugar-phosphate backbone modifications on target DNA cleavage by T. thermophilus Ago. Cleavage experiments were performed as described in Methods. a, 2'-fluoro-, 2'-methoxy- and 2'-hydroxyl-substitutions of single 2'-deoxyribose residues of the target DNA strand at and near the cleavage site. The control target (unmod.) was the unmodified oligodeoxynucleotide. b, Phosphorothioate modification of the target DNA. The phosphate configuration (R[P] or S[P]) of the phosphorothioate diastereomers is indicated. Cleavage assays were performed in the presence of either Mg^2+ or Mn^2+ cations. Note that the experiment for the 11'–12' isomers was a different experiment, in which overall reaction rates were slower. For the complete experiment see Supplementary Fig. 25. Sequences of oligonucleotides are in Supplementary Table 4. c, Structure of the cleavage site modelling the attack of the hydroxyl nucleophile. Phosphate oxygen and active site carboxylate oxygens coordinated to metal ions A and B (purple spheres), with distances less than 2.5 Å shown as blue dashed lines. The coordination of the carboxylate oxygen from Asp 546 to metal ion B is hidden in the projection. The phosphate oxygens and 2' residues sensitive to modification are shown as yellow and green spheres, respectively; R denotes 2'-H, -OH, -F or -Ome. Red arrows indicate the attack of the hydroxyl nucleophile modelled to be directly coordinated by metal ion A, and the stabilization of the developing negative charge of the 3' oxyanion leaving group by metal ion B.

PROCHECK

	Headers