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.
How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-β-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.
Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand. Expected final online publication date for the Annual Review of Virology, Volume 7 is September 29, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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.
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.
Cancer (COSMIC) database and mapped on the TRPA1 structure. TRPA1 structures harbouring OAC mutations were simulated in order to inform alterations in structure-function relationships of the channel and propose ways by which mutations promote cancer. Notably, we observe clustering of cancer mutations around dynamic hinges in the intracellular ankyrin repeat region of the structure. We propose that missense mutations observed in OAC promote cancer via TRPA1 activation by altering protein dynamics. Increasing evidence suggests that ion channels are not only responsible for regulating cellular homeostatic mechanisms such as cell volume, membrane potential, and osmolarity but they are also implicated in controlling cell proliferation, migration, apoptosis, and differentiation. Aberrant cell cycle control, cell adhesion, and migration contribute to an invasive phenotype associated with cancer and metastasis. Recent evidence illustrates that disruptions in calcium signalling, secondary to the overactivity of TRPA1, promote Reactive Oxygen Species (ROS) tolerance and apoptosis resistance in cancer cells.We have previously shown that TRPA1 is as a driver gene and is recurrently mutated in 6% of a 551 OAC-patient cohort based on the ratio of nonsynonymous to synonymous mutations. Here we confirm this through a detailed analysis of patient data, as well as the behaviour and properties of missense mutations along the protein structure. Kaplan-Meier survival curves from The Cancer Genome Atlas (TCGA) and Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) datasets, reveal that high TRPA1 expression correlates with poor patient prognosis in OAC (p = 0.019), and observe changes in canonical cancer pathways with TRPA1 expression across the cohort.
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