PDBsum entry 3f73

Nucleic acid binding protein/DNA/RNA

PDB id: 3f73

Contents

Protein chains

678 a.a. *

DNA/RNA

Ligands

PO4

Metals

_MG ×3

Waters ×16

* Residue conservation analysis

PDB id:

3f73

Name:

Nucleic acid binding protein/DNA/RNA

Title:

Alignment of guide-target seed duplex within an argonaute silencing complex

Structure:

Argonaute. Chain: a, b. Engineered: yes. DNA (5'- d(p Dtp Dgp Dap Dgp Dgp Dtp Dap Dgp Dtp Dap Dgp Dgp Dtp Dtp Dgp Dtp D a Dtp Dap Dgp Dt)-3'). Chain: c, x. Engineered: yes. RNA (5'-

Source:

Thermus thermophilus. Organism_taxid: 262724. Strain: hb27. Gene: tt_p0026. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic: yes

Resolution:

3.00Å R-factor: 0.221 R-free: 0.282

Authors:

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

Key ref:

Y.Wang et al. (2008). Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature, 456, 921-926. PubMed id: 19092929 DOI: 10.1038/nature07666

Date:

07-Nov-08 Release date: 16-Dec-08

Protein chains

Q746M7  (Q746M7_THET2) -  Protein argonaute from Thermus thermophilus (strain ATCC BAA-163 / DSM 7039 / HB27)

Seq: 685 a.a.

Struc: 678 a.a.

DNA/RNA chains

T-G-A-G-G-T-A-G-T-A-G-G-T-A-G-T 16 bases

C-U-C-A-C-U-A-C-C-U-C-G 12 bases

T-G-A-G-G-T-A-G-T-A-A-T-A-G-T 15 bases

U-C-A-C-U-A-C-C-U-C-G 11 bases

DOI no: 10.1038/nature07666

Nature 456:921-926 (2008)

PubMed id: 19092929

Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex.

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

ABSTRACT

Here we report on a 3.0 A 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 formation 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.

Selected figure(s)

Figure 1.
Figure 1: Crystal structure of T. thermophilus Ago bound to 5'-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA. a, Sequence of the guide DNA-target RNA duplex. The traceable segments of the bases of the guide DNA and target RNA in the structure of the ternary complex are shown in red and blue, respectively. Disordered segments of the bases on both strands that cannot be traced are shown in grey. b, Stereo view of the 3.0 Å crystal structure of the Ago ternary complex. The Ago protein is colour-coded by domains (N in cyan, PAZ in magenta, Mid in orange and PIWI in green) and linkers (L1 and L2 in grey). The bound 21-nucleotide guide DNA is in red and traced for bases of the 1–10 and 19–21 segments, whereas the bound 20-nucleotide target RNA is in blue and traced for bases of the 1' to 9' segment. Backbone phosphorus atoms are in yellow. c, An alternate view of the complex.

Figure 2.
Figure 2: Comparison of structural details between the binary Ago complex with bound guide DNA and the ternary complex with added target RNA. a, Expanded view of the ternary complex highlighting the guide DNA (1–10)-target RNA (1'–9') duplex and Mg^2+-coordinated catalytic residues (D478, D546 and D660) of the RNase H fold of the PIWI domain. Intermolecular hydrogen bonds between the Ago protein and the DNA guide strand in red are shown by dashed lines. b, Positioning of the sugar-phosphate backbone of the target RNA strand spanning the mismatch-containing 10–11-step relative to the catalytic residues of the PIWI domain. c, Comparison of the trajectory of traceable bound guide DNA in the binary (bases 1–11 and 18–21 in silver) and ternary (bases 1–10 and 19–21 in red) Ago complexes after superposition of their 5'-phosphate-binding pockets. d, Superposition of the guide DNA (red)-target RNA (blue) duplex spanning the 2–8 seed segment on A-form (left panel) and B-form (right panel) helices (silver) after best-fit superposition of the target RNA strand of the ternary Ago complex with one strand of the A/B-form helices. e, Positioning of stacked residues 6–10 of the DNA guide strand relative to R548, with emphasis on intermolecular interactions involving the sugar-phosphate backbone. f, Relative positioning of the 6 to 10/11 segment of the bound guide DNA strand and R548 in the binary (guide strand in silver, protein in cyan) and ternary (guide strand in red, protein in magenta) Ago complexes. The conformational change in the protein on proceeding from binary to ternary Ago complexes is indicated by a red arrow.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: Nature (2008, 456, 921-926) copyright 2008.

Figures were selected by an automated process.