Identification of the compendium of factors interacting with translating ribosomes in E.coli

Kumar, Sneha (2017): Identification of the compendium of factors interacting with translating ribosomes in E.coli: a novel proteomic approach. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

How newly synthesized proteins fold to their functionally active conformations remains one of the fundamental questions in biology. Studies in vivo and in vitro, conducted over the last decade, indicate that translation and folding are coupled. Various components, such as molecular chaperones, proteases and other factors, interact with nascent polypeptide chains emerging from ribosomes and assist in nascent chain folding, quality control, subcellular targeting and co-translational modifications. A complete compendium of these factors is still missing and our understanding of the cellular machinery that acts co-translationally on the ribosome is still incomplete. In this study we sought to identify the factors that interact with translating ribosomes in E. coli cells using quantitative proteomics methods. Antibiotics, which inhibit translation in bacteria by interfering with protein synthesis at specific stages, were employed to generate ribosome nascent chain complexes amenable to isolation and unbiased analysis. Two examples of antibiotics used in this study are chloramphenicol and puromycin. Both act on the ribosome by targeting the ribosomal peptidyl transferase center. By combining translation inhibition with chemical crosslinking in spheroplasts and analysis by SILAC-based mass spectrometry, we designed a method that can be used to generate an inventory of factors contributing towards efficient folding of nascent chains in the cellular environment. We identified approximately 378 factors that interact specifically with translating ribosomes and are depleted upon nascent chain release from the ribosome. These factors could be divided into the following main categories: Molecular chaperones, RNA quality control proteins, transcription factors, translation factors and translocation factors. Chaperones ofthe small heat shock protein family, disaggregates and proteases, apparently interact with nascent chains in a concerted manner to support productive folding, reverse misfolding and remove aberrant protein chains. Various transcription factors also interacted substantially with ribosome nascent chain complexes (RNCs), suggesting their additional function in translation and folding. Our analysis of RNCs provides further insights into the mechanism of co-translational translocation and helps to elucidate the role of chaperones and translocon protein complex in guiding co-translational targeting and stabilizing secretory and membrane proteins. The data provide a valuable resource for future functional studies.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Chaperones, Ribosomes , Nascent Chains
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 18. Januar 2017
• 1. Bericht­erstatter:in: Hartl, F. Ulrich