Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

The S. cerevisiae strain YAT547 (kar1) containing pGKL plasmids (42) and isogenic S. cerevisiae ρ 0 strains bearing the wild-type CDC33 gene (CWO4ρ 0 CDC33wt) and its temperature-sensitive mutations (CWO4ρ 0 cdc33-1 and CWO4ρ 0 cdc33-42, for exact . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/325316 doi: bioRxiv preprint genotypes see Table S3 ) were cultured separately in YPD medium at 24°C overnight. Cells were resuspended the next day in 200 µl of water, and 5 μl from each of two required strains (approximately 3x10 6 cells) were mixed on the YPD agar plate and further incubated for 5 hours at 24°C. Cells were then scraped to liquid YPD medium, incubated at 24°C with gentle shaking overnight and plated on selective plates. The resulting strains were tested for their auxotrophic markers, mating type and for killer toxin production under both permissive and non-permissive conditions. Strains showing a temperature-sensitive phenotype (when applicable), killer toxin production, growth on SD medium lacking methionine, no visible growth on SD medium lacking uracil and a MATa mating type were used for subsequent analyses.