Supplementary Fig. 6: Isolation and Vms1p cleavage of RNCs with radiolabeled peptidyl-tRNALeu.
a, TBE-urea-PAGE and SYBR Gold (top) or autoradiography (bottom) analysis of tRNALeu transcribed with either radiolabeled CTP or UTP. b, Purification of stalled 80S ribosome-nascent chain complexes (RNCs) from cell-free translation reactions of the truncated mRNA depicted in Fig. 2a with either C- or U-labeled tRNALeu. IVT—total in vitro translation reaction; HS-RNCs—high-salt-washed RNCs; FT—flow-through; Elu—elution. c, 35S-methionine labeled RNCs generated using the same model mRNA as in b with total liver tRNAs were incubated with energy, 50–100 nM ribosome splitting factors, and 10 nM NEMF and 125 nM wildtype (WT) or R288A (mut.) Vms1p as indicated. Reactions were directly analyzed before or after RNase A treatment by NuPAGE and autoradiography. This demonstrates that the requirements for Vms1p activity on RNCs containing peptidyl-tRNALeu are identical to those containing peptidyl-tRNAVal (Fig. 1). d, Coomassie stain of the SDS-PAGE gel analyzed in Fig. 2b. Note that 20-fold less of the reactions containing 35S-methionine-labeled RNCs were loaded to roughly normalize the autoradiography signals. e, Radiolabeled tRNALeu at approximately one (1X) or five (5X) times the concentration isolated from RNCs (for example Fig. 2c) was incubated with 125 nM Vms1p without or with an energy regenerating system (ERS). Without being incorporated into RNCs, there is no noticeable Vms1p cleavage of free tRNAs. Transcribed tRNALeu with (FL) or without (ΔCCA) the 3′CCA nucleotides are loaded as size markers.
