Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning

Carmody, Patrick J.; Zimmer, Matthew H.; Kuntz, Charles P.; Harrington, Haley R.; Duckworth, Kate E.; Penn, Wesley D.; Mukhopadhyay, Suchetana; Miller, Thomas F.; Schlebach, Jonathan P.

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. In the alphavirus structural polyprotein, -1PRF is coordinated by a “slippery” sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.