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

On days of binding experiments, bacterial lysate containing GST-eIF4E fusion protein was thawed in an ice bath, centrifuged for 10 min at 15000 g (4°C), and GST-eIF4E was purified using GST-affinity chromatography in a batch setup. In brief, 1 ml of the lysate was incubated for 80 min with 100 μl of glutathione-Sepharose 4 Fast Flow resin (GE Healthcare) at 4°C and washed four times with at least 40 volumes of ice-cold 1x phosphate-buffered saline (PBS). After the last washing step, the glutathione Sepharose binding GST-eIF4E fusion protein was resuspended in 200 µl of buffer I (20 mM HEPES, pH 7.5; 0.1 mM EDTA, pH 8.0; 100 mM KCl; and 1 mM β-mercaptoethanol) and mixed with 3 µg of DNase I-treated total RNA purified from K. lactis IFO1267. Next, 2 µl of the reaction mixture was removed for subsequent analysis using RT-PCR and real-time quantitative PCR (RQ-PCR). The rest of the mixture was incubated for two hours at room temperature and washed six times (with approximately 70 volumes) with buffer I. After the last washing, the glutathione Sepharose binding GST-eIF4E fusion protein and RNA and control samples were subjected to RT-PCR.