FIGURE 2.

Mapping of the stall sites of arrested and stacked ribosomes. A, nucleotide and amino acid sequences around the MTO1 region. Synonymous codon substitutions were generated at the sites marked. B–E, SDS-PAGE analysis of the translation products of M8:Ex1 RNA (50 fmol μl⁻¹) in the absence (−) or presence (+) of 1 mm AdoMet. The AdoMet-dependent PtR and the full-length translation product (FL) are marked. PtR(a), blue circle; PtR(b), magenta circle; PtR(c), green circle. Representative results of triplicated experiments are shown. B, effects of synonymous substitutions at the Ser-94 codon. PtR(b1) (magenta triangle) and PtR(b2) (magenta square) bands are marked. C and D, The Val-85 codon (C) and the Gly-84 and Ile-83 codons (D) were synonymously substituted. PtR(a1) (blue triangle) and PtR(a2) (blue square) bands are marked in C. E, the codons from Ile-74 to Arg-77 were synonymously substituted. PtR(c1) (green triangle) band is marked. F, schematic representation of M8:ND5(C80A) RNA. The region of the CGS1 exon 1 that was absent in the ND5 deletion (25) is indicated by a dashed line. The Cys-80 to alanine substitution is shown (C80A). G, cysteine-scanning experiment to detect [³⁵S]cysteine-labeled PtRs. M8:ND5(C80A) RNAs (50 fmol μl⁻¹) carrying only one cysteine codon at the indicated amino acid positions were translated in the presence of 1 mm AdoMet and subjected to SDS-PAGE analysis. Positions of the AdoMet-dependent PtR and the full-length translation product (FL) are marked. C80C carries the original cysteine codon. A representative result of duplicated experiments is shown. H, scanned images of E, lane 2, and G, lane 2 are presented.