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 mRNA 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 its 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 dynamics 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 indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.

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

“…3). Consistent with previous findings (Harrington et al, 2020), our results show that the introduction of polar or charged side chains within a certain region of TM2 reduces -1PRF (V735-V746) while mutations that enhance the hydrophobicity of TM2 generally increase -1PRF (Fig. 3).…”

“…Our recent investigations of the impact of the nascent chain on -1PRF revealed that the mechanical force generated by the translocon-mediated membrane integration of the second TM domain within the E2 protein enhances ribosomal frameshifting in the SINV structural polyprotein (Harrington et al, 2020). Our DMS results reveal that ribosomal frameshifting is sensitive to a variety of mutations that alter the amino acid sequence within the portion of the nascent chain that has been translated at the point of frameshifting, including mutations to residues within the exit tunnel, between the exit tunnel and translocon, and within the second transmembrane domain of E2 (TM2, Fig.…”

“…Frameshifting is also enhanced by the depletion of the t-RNA pool that decodes the YYZ 7 codon (“hungry” frameshifting), which provides the opportunity for the P-site tRNA to move out of frame (Korniy et al, 2019b). In addition to mRNA and tRNA effectors, our group recently found that -1PRF is enhanced by mechanical forces generated by the cotranslational folding of the nascent polypeptide chain (Harrington et al, 2020). This finding has since been echoed by recent evidence suggesting cotranslational folding of nsp10 alters -1PRF during translation of SARS-CoV-2 genome (Bhatt et al, 2020).…”

Residue-by-residue analysis of cotranslational membrane protein integration in vivo

“…A detailed view of the cotranslational integration of three multispanning membrane proteins provided here shows that translocating nascent chains experience a distinct transition to a more hydrophobic environment at a distance of ~45 residues from the PTC, generating an oscillating force on the nascent chain that is ultimately transmitted to the PTC and varies in step with the appearance of each TMH in the vicinity of the SecYEG translocon channel. It seems likely that such oscillations can have multiple effects on the translation of membrane proteins, as recently demonstrated for ribosomal frameshifting ( Harrington et al, 2020 ), and may affect protein quality control ( Lakshminarayan et al, 2020 ).…”

Residue-by-residue analysis of cotranslational membrane protein integrationin vivo

“…The high-resolution view of the cotranslational integration of multi-spanning membrane proteins provided here show that translocating nascent chains experience a distinct transition to a more hydrophobic environment at a distance of ~45 residues from the PTC, generating an oscillating force on the nascent chain that is ultimately transmitted to the PTC and varies in step with the appearance of each TMH in the vicinity of the SecYEG translocon channel. It seems likely that such oscillations can have multiple effects on the translation of membrane proteins, as recently demonstrated for ribosomal frameshifting (25), and may affect protein quality control (26). Charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a TMH can advance, delay, or attenuate its interaction with the translocon and surrounding lipid.…”