A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery [preprint]
AuthorsNg, Martin Y.
Jamiolkowski, Ryan M.
Cooperman, Barry S.
UMass Chan AffiliationsDepartment of Microbiology and Physiological Systems
eukaryotic protein synthesis machinery
single molecule FRET
Amino Acids, Peptides, and Proteins
Nucleic Acids, Nucleotides, and Nucleosides
MetadataShow full item record
AbstractNonsense suppressors (NonSups) treat premature termination codon (PTC) disorders by inducing the selection of near cognate tRNAs at the PTC position, allowing readthrough of the PTC and production of full-length protein. Studies of NonSup-induced readthrough of eukaryotic PTCs have been carried out using animals, cells or crude cell extracts. In these studies, NonSups can promote readthrough directly, by binding to components of the protein synthesis machinery, or indirectly, by inhibiting nonsense-mediated mRNA decay or by other mechanisms. Here we utilize a highly-purified in vitro system (Zhang et al., 2016. eLife 5: e13429) to measure exclusively direct NonSup-induced readthrough. Of 17 previously identified NonSups, 13 display direct effects, apparently via at least two different mechanisms. We can monitor such direct effects by single molecule FRET (smFRET). Future smFRET experiments will permit elucidation of the mechanisms by which NonSups stimulate direct readthrough, aiding ongoing efforts to improve the clinical usefulness of NonSups.
bioRxiv 330506; doi: https://doi.org/10.1101/330506. Link to preprint on bioRxiv service.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/29272
Related ResourcesNow published in ACS Medicinal Chemistry Letters doi: 10.1021/acsmedchemlett.8b00472
RightsThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
Except where otherwise noted, this item's license is described as The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.