Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock

Author:

Giudice Emmanuel1,Georgeault Sylvie1,Lavigne Régis23,Pineau Charles23,Trautwetter Annie1,Ermel Gwennola1,Blanco Carlos1,Gillet Reynald1

Affiliation:

1. Univ Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR6290, 35000 Rennes, France

2. Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail)-UMR_S 1085, 35000 Rennes, France

3. Univ Rennes, CNRS, Inserm, Biosit UAR 3480 US_S 018, Protim Core Facility, 35000 Rennes, France

Abstract

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

Funder

Agence Nationale pour la Recherche (ANR)/Direction Générale de l’Armement

Fondation pour la Recherche Médicale

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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