Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

COORDINATION OF -1 PROGRAMMED RIBOSOMAL FRAMESHIFTING BY THE TRANSCRIPT AND NASCENT POLYPEPTIDE Patrick Carmody 1 ; Matthew Zimmer; Francis J Roushar 1 ; Dyotima Dyotima 1 ; Haley R Harrington 1 ; Wesley D Penn 1 ; Thomas F Miller III; Suchetana Mukhopadhyay 1 ; Jonathan Schlebach 1 ; 1 Indiana University, Bloomington, Chemistry, Bloomington, IN, USA Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism involved in the regulation of protein biosynthesis. PRF is stimulated by RNA structures that pause the ribosome during the decoding of a “slippery” sequence that allows the tRNA to slip into alternative reading frames. We recently showed that the cotranslational folding of the alphavirus structural polyprotein at the endoplasmic reticulum membrane generates pulling forces on the ribosome that enhance the efficiency of -1PRF. To explore this mechanochemical feedback, we measured the effects of 4,530 mutations on the efficiency of -1PRF by deep mutational scanning. Mutational trends near the slip-site reveal the sequence constraints within stimulatory RNA structures while trends upstream of the site suggest frameshifting can be tuned by the interaction of the nascent chain with the ribosome and translocon. Molecular modeling and coarse-grained molecular dynamics simulations suggest forces are generated by the formation of a translocation intermediate that forms within the translocon. These findings demonstrate that the activity of the ribosome can respond to cotranslational folding in real time. As an extension of these studies, we show that similar -1PRF sites can be engineered into other transcripts and carry out an informatic analysis to identify putative frameshift motifs in the human transcriptome. Finally, we validate a novel -1PRF site within the cystic fibrosis transmembrane conductance regulator (CFTR) and provide preliminary evidence to suggest that it may function as a quality control checkpoint during translation. Together these findings reveal how structural features within the transcript and nascent chain coordinate translational regulation.

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