FIGURE 5.

Effect of exogenously added peptide covering the MTO1 region on puromycin reaction. A, amino acid sequences of the synthetic peptides carrying wild-type MTO1 region (upper panel) and that with mto1–1 mutation (lower panel). The mto1–1 mutation is marked in red. To prevent dimerization, a Cys-80 to alanine substitution was introduced (marked in blue), which is among the few substitutions that are tolerated within the MTO1 region (25). B, M8:Ex1(WT) RNA (50 fmol μl⁻¹) was translated in the presence of 1 mm AdoMet with 500 nm synthetic peptide carrying wild-type MTO1 sequence or mto1–1 mutation, or without synthetic peptide (mock). After 30 min of translation, puromycin was added to a final concentration of 2 mm. Samples were removed for SDS-PAGE before (0 min) and 1 min after the start of puromycin reaction. The AdoMet-dependent PtRs, the full-length translation product (FL) and peptidyl-puromycin (PP) bands are marked. A representative result of triplicated experiments is shown. C, radioactive signals in B were quantified. The intensity of unreacted PtR was normalized to that at time 0. Average ± S.D. (n = 3) are shown.