Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

Huter, Paul (2018): Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Ribosome, Translation, ArfA, ArfB, tmRNA, SmpB, EF-P, Prolines
• 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: 20. Juni 2018
• 1. Bericht­erstatter:in: Wilson, Daniel