Biophysical effects of the conformational flexibility of a viral frameshiftelement on local RNA tension

Ribosomal frameshifting is a critical moment in many coronaviruses' life cycles. In this process, an untranslated mRNA structure called the frameshift element (FSE) stalls the ribosome over an upstream "slippery" site, allowing the ribosome to shift 1 nucleotide back into a new reading frame. FSE frameshifting efficiency is tailored from virus to virus to produce the correct ratio of protein expression in the initial and shifted reading frames. Because of this, disruption of frameshifting is a key future target for antiviral development.

The efficiency of a given FSE depends not on the stability of a single folded structure, but on the structural heterogeneity of the sequence. However, the mechanism by which conformational dynamics affect frameshifting efficiency is unclear.

In this project, we elucidate this mechanism by investigating the FSE from SARS-CoV-2 using magnetic tweezers with single-molecule FRET. This FSE, which has 15-30% frameshifting efficiency can assume multiple conformations, including an unusual threaded configuration. We use magnetic tweezers to identify whether the FSE is in a folded or unfolded state and FRET to monitor the local tension in the slippery site. From these observations, we will determine whether wrapping of the linker RNA into the threaded state produces tension fluctuations significant enough to disrupt ribosome-RNA interactions and induce frameshifting, as has been suggested.