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

Abstract

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.

Stepwise evolution and exceptional conservation of ORF1a/b overlap in coronaviruses

“…Our results provide an alternative way to assess exceptional conservation of the PFE using publicly available sequence data highlighting the fact that the entire PFE region appears to be under strong purifying selection. These patterns are similar to observations obtained from deep mutational scanning where any alteration at the majority of PFE region sites results have deleterious effects on the frameshift efficiency (e.g., (Carmody et al 2021)).…”

“…The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.14.448413 doi: bioRxiv preprint where any alteration at the majority of PFE region sites results have deleterious effects on the frameshift efficiency (e.g., (Carmody et al 2021)).…”