Abstract. Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. A
. The reaction was stopped by dilution into twenty volumes of TriPURE isolation reagent. Subsequent phenol-extraction was performed according to the manufacturer's instructions. The pep-tRNAs in the aqueous phase were precipitated by isopropanol. The pep-tRNAs in the organic phase were precipitated by TCA/acetone precipitation as described above.
was added and the lysate was centrifuged at 12 000 × g for 3 min at 4°C to remove the precipitates. The supernatant was loaded into 4 columns, and the following steps were performed according to the manufacturer's instructions. The silica-bound RNA was eluted with 100 μl of PTS buffer, and the collected eluate was divided into two equal portions, and one was treated with 100 μg/ml of RNase A at 37°C for 1 h. This purification was repeated twice.
The measurement for the comparison against the phenol-extracted proteins was performed in DDA mode using a Q-Exactive tandem mass spectrometer and an Easy-nLC 1000 nano HPLC system . The detailed settings were as described above. The data analysis was performed using Proteome Discoverer 2.4 with the same settings as described above.
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