To prepare take-off complexes, translation mixtures containing 70S ribosomes or fluorescence-labeled 30S subunits together with a 1.5-fold excess of labeled 50S subunits (0.16 μM) were incubated for 20 min at 10°C, and the resulting ribosome complexes were purified by gel filtration on BioSuite 450 HR 8-μm column (Waters) at 4°C. The amount of the nascent peptide bound to the ribosome was calculated from the ratio f[3H]Met-tRNAfMet radioactivity to OD260 in the ribosome fraction.

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