title: Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex.
abstract: Trax-translin heteromers, also known as C3PO, have been proposed to activate the RNA-induced silencing complex (RISC) by facilitating endonucleolytic cleavage of the siRNA passenger strand. We report on the crystal structure of hexameric Drosophila C3PO formed by truncated translin and Trax, along with electron microscopic and mass spectrometric studies on octameric C3PO formed by full-length translin and Trax. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoRNase activity and interacts extensively with translin to form an octameric assembly. The catalytic pockets of Trax subunits are located within the interior chamber of the octameric scaffold. Truncated C3PO, like full-length C3PO, shows endoRNase activity that leaves 3'-hydroxyl-cleaved ends. We have measured the catalytic activity of C3PO and shown it to cleave almost stoichiometric amounts of substrate per second.
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.forename: Dhirendra K
authors.initials: DK
authors.lastname: Simanshu
authors.forename: Manuel
authors.initials: M
authors.lastname: Ascano
authors.forename: Ruben
authors.initials: R
authors.lastname: Diaz-Avalos
authors.forename: Ah Young
authors.initials: AY
authors.lastname: Park
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Juranek
authors.forename: William J
authors.initials: WJ
authors.lastname: Rice
authors.forename: Qian
authors.initials: Q
authors.lastname: Yin
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000201:enzymology*; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012313:RNA / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C542957:Trax protein, Drosophila; C509504:trsn protein, Drosophila; D012313:RNA; D004722:Endoribonucleases