Abstract
Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that 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.
Highlights
Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that occurs in all kingdoms of life
To probe the sequence constraints of −1PRF, we employed deep mutational scanning (DMS) to map the effects of mutations on ribosomal frameshifting within the Sindbis virus (SINV) structural polyprotein
Mutagenic effects were assessed in the context of a reporter that selectively generates a fluorescent protein as a result of −1PRF during translation and processing of the SINV structural polyprotein (Figure 1A)
Summary
Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that occurs in all kingdoms of life. Viruses utilize ribosomal frameshifting to increase their genomic coding capacity and to regulate the stoichiometric ratios of viral protein synthesis. Some rely on these motifs to coordinate genomic replication, while others utilize PRF to regulate the production of the structural proteins that mediate assembly [2]. The frameshift products are themselves virulence factors that antagonize the host interferon response [3,4,5,6] For these reasons, the efficiency of PRF, which is globally regulated by both host and viral proteins [7,8], is often critical for infection and immunity
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