Multimeric assembly and biochemical characterization of the Trax - translin endonuclease complex

• Published in: Nature structural & molecular biology Vol. 18; no. 6; pp. 658 - 664
• Main Authors: Tuschl, Thomas, Patel, Dinshaw J, Tian, Yuan, Simanshu, Dhirendra K, Ascano, Manuel, Diaz-Avalos, Ruben, Park, Ah Young, Juranek, Stefan A, Rice, William J, Yin, Qian, Robinson, Carol V
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-06-2011; Nature Publishing Group
• Subjects: 631/337/1645; 631/45/535; Animals; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Catalytic Domain; CLEAVAGE; CRYSTAL STRUCTURE; Crystallography, X-Ray; DROSOPHILA; Drosophila melanogaster - chemistry; Drosophila melanogaster - enzymology; Drosophila Proteins - chemistry; Drosophila Proteins - metabolism; ELECTRONS; ENDONUCLEASES; Endoribonucleases - chemistry; Endoribonucleases - metabolism; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; Kinetics; Life Sciences; Mass Spectrometry; Membrane Biology; Microscopy, Electron; Models, Molecular; Molecular biology; OCCUPANTS; Protein Multimerization; Protein Structure; Protein Structure, Quaternary; Proteins; Ribonucleic acid; RNA; RNA - metabolism; SUBSTRATES; United States
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.2069

C3PO was originally identified in
Drosophila
as a complex that enhances RNA-induced silencing complex (RISC) activity. EM and X-ray crystal structures of the
Drosophila
C3PO complex are now presented and endoribonuclease activity characterized.
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.