Sidhu, Haneesh Kaur; (2021) Dissecting the role of the Ribosome in Cotranslational Folding using CRISPR-Cas9. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The ribosome is the universal orchestrator of protein synthesis and in vivo many proteins begin to fold cotranslationally while they are being synthesised on the ribosome. Emerging evidence suggests that the ribosome plays an active role to influence the folding pathway and prevent misfolding. Burrowing through the exit tunnel to the ribosome surface, the nascent chain experiences a unique environment. Here we apply CRISPR-Cas9 genome editing to dissect the influence of the ribosomal exit tunnel proteins on cotranslational folding. The ribosomal protein loops of uL4, uL22, uL23 and uL24 contribute to the geometry of the tunnel and are highly conserved yet display some interesting evolutionary differences across the three domains of life. These protein loops necessarily interact with the diverse range of nascent polypeptides in the proteome. We rationally designed and generated a series of ribosomal variants with altered tunnel geometries via truncations and insertions in ribosomal protein tunnel loops. An integrated approach was used to characterise ribosomal variants combining biochemical assays, NMR spectroscopy, cryo-EM, and molecular dynamics simulations. Cotranslational folding of an immunoglobulin-like domain, the filamin FLN5, can be both enhanced or inhibited with different ribosomal loop variants, with the loops of uL23 and uL24 in the vestibule having a pronounced effect. In depth investigation of the mechanistic basis for earlier folding of these ribosomal variants is presented, allowing us to define the modulation of the free energy landscape for folding. This study is the first systematic application of CRISPR-Cas9 genome editing to the ribosomal exit tunnel and thus contributes to understanding key questions regarding the earliest encounters of newly synthesising polypeptides, specifically how the geometry of the exit tunnel influences the folding of the nascent chain and modulates its folding pathway.

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