Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Abstract

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamic simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip-site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results demonstrate that cotranslational folding of this protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding and/ or binding events may also stimulate PRF.

The SARS-CoV-2 Programmed −1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography

“…However, during translation with translocating ribosomes upstream and downstream, the PFSE region would be unfolded and isolated from the rest of the genomic RNA enabling formation of the frameshifting structure. 4,17,46 Additionally, the finding that the MTDB can inhibit viral replication and alters the ratios of the conformations the PFSE can adopt suggests that structures adopted by the minimal PFSE element have relevance for viral function. 4,5 Comparison of our Crystal Structure to cryo-EM Structures.…”

“…4,13,19 The basic mechanism of −1 ribosomal frameshifting is known, although there are many levels of regulation at play that are not understood. 17,[20][21][22]46 Generally, a structured region of the RNA causes a translating ribosome to pause over a so-called slippery site with a nucleotide sequence pattern of X XXY YYZ composition. 16,18,23,24 This structured region is most often a pseudoknot, which forms 6−8 nts downstream of the slippery site.…”

Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon

“…Measuring these interactions and determining which ones are dominant at different stages of translation is challenging due to the complexity of the co-translational environment. Yet, an understanding of the molecular interactions that govern co-translational processing via the Sec translocon is essential for the design of modifications that alter the outcome of this process . A promising method for measuring co-translational forces in an in vivo environment relies on arrest peptides (APs) which stall translation at a precisely definable location .…”