The elongation and termination assays were performed as previously described (38 (link)) using MFFFKX-UAA initiation complexes or MFX-UAA pre-termination complexes with the addition of 10–20 μM Upf1 ΔCH.
The recycling assay was performed using MFKX-UAA pre-termination complexes in reaction with 1X Buffer E, 4 μM AGQ-eRF1, 2 μM Rli1 and 10 μM Upf1 ΔCH. 50 μM peptidyl-tRNA hydrolase (PTH) was added to the reactions to quantify dissociated complexes (PTH cannot access the peptidyl-tRNA unless released from the ribosome) (46 (link)). Time points in both assays were quenched using 10% formic acid and run on electrophoretic TLC (Millipore).
All TLC plates were developed using a Typhoon FLA 9500 Phosphorimager system and quantified using ImageQuantTL (GE Healthcare Life Sciences). Time courses were fit to single exponential kinetics using Kaleidagraph (Synergy Software).
The recycling assay was performed using MFKX-UAA pre-termination complexes in reaction with 1X Buffer E, 4 μM AGQ-eRF1, 2 μM Rli1 and 10 μM Upf1 ΔCH. 50 μM peptidyl-tRNA hydrolase (PTH) was added to the reactions to quantify dissociated complexes (PTH cannot access the peptidyl-tRNA unless released from the ribosome) (46 (link)). Time points in both assays were quenched using 10% formic acid and run on electrophoretic TLC (Millipore).
All TLC plates were developed using a Typhoon FLA 9500 Phosphorimager system and quantified using ImageQuantTL (GE Healthcare Life Sciences). Time courses were fit to single exponential kinetics using Kaleidagraph (Synergy Software).
