Fiche publication
Date publication
janvier 2020
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr SIMONETTI Angelita
Tous les auteurs :
Rol-Moreno J, Kuhn L, Marzi S, Simonetti A
Lien Pubmed
Résumé
Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.
Mots clés
Cryo-electron microscopy, Mass spectrometry, Native ribosome complexes, Translation initiation
Référence
Methods Mol. Biol.. 2020 ;2113:329-339
