Fig. S4.

In vitro analyses of VemP protein. (A) Calibration of the VemP toeprint signal. The vemP gene without (lanes 1 and 2) or with (lane 3) a missense mutation or a nonsense mutation as indicated (lanes 4–9) was subjected to the PURE system reaction that lacked the E. coli ribosome (−) (lane 2) or that was supplemented with ribosome prepared from V. alginolyticus (Va ribo) (+) (lanes 1 and 3–9). Additionally, the release factors 1, 2, and 3 were omitted for lanes 4–9 to make the ribosome stall at the defined stop codons. The translation complexes were then used as a template for the reverse transcriptase reaction that was primed by a downstream primer as described in SI Materials and Methods. The cDNA fragments were separated by 6% polyacrylamide–7 M urea gel along with the dideoxy sequencing ladders (left four lanes). Comparison of the signal produced from WT vemP (asterisk, lane 1) with those of the control samples (lanes 4–9) allowed us to identify the point of VemP translation arrest, where the Phe157 codon resides in the A site of the ribosome. (B) Western and Northern blotting characterization of VemP′-tRNAs. The flag-vemP fusion gene, encoding the Flag epitope tag followed by residues 26–159 of VemP (lanes 1 and 5), and flag-vemP with the F157stop mutation (lanes 4 and 8) were subjected to in vitro translation reaction using the Vibrio hybrid PURE System (lanes 1–4) or E. coli PURE System (lanes 5–8) in the presence of release factors. As controls we also used flag-vemP mRNA truncated after the 156th codon (lanes 2 and 6) and its Q156F version (lanes 3 and 7) as a template. The products were separated by 10% Nu-PAGE and the regions, where VemP′-tRNA molecules migrated, were analyzed by anti-FLAG immunoblotting (Upper) and Northern blotting using specific probes for tRNA^Gln (Middle) and tRNA^Phe (Lower). WT VemP′-tRNA was hybridized with the tRNA^Gln probe (lane 1), indicating that the arrested nascent product contained a tRNA^Gln moiety. Thus, VemP translation undergoes an arrest at the step of peptidyl transfer from VemP_1–156-tRNA^Gln to the A-site Phe-tRNA^Phe. The product from the vemP(F157Stop) mutant was also hybridized with the same probe, indicating that the nascent VemP can arrest the termination step of translation as well. (C) Determination of the C-terminal end required for the VemP translation arrest. PCR products of vemP with a stop codon placed at the positions indicated were used as a template for in vitro translation in the presence of [³⁵S]methionine for 45 min at 37 °C. The samples, treated without (lanes 1–7) or with (lanes 8–14) RNase A, were separated by 10% neutral SDS/PAGE, followed by phosphor imaging. (D) Effects of VemP amino acid substitutions on efficiency of translation arrest. WT and mutant forms of vemP having the indicated amino acid substitution mutations were translated using the Vibrio hybrid PURE system (the upper gel) and E. coli PURE system (the lower gel) in the presence of [³⁵S]methionine at 37 °C for 45 min. Synthesized proteins were separated by neutral 10% SDS/PAGE. Proportions (percent) of the arrested VemP-tRNA band in the sum of the tRNA-less VemP and the VemP-tRNA bands are presented with SD in Fig. 4E (n ≥ 2 in the upper panel). Amino acid residues whose mutations compromised the translation arrest strongly and less strongly are highlighted in red and blue, respectively, at the top.