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. 2022 Jun 11;298(7):102133. doi: 10.1016/j.jbc.2022.102133

Figure 1.

Figure 1

An approach for determining the level of stop codon readthrough in the reconstituted eukaryotic translation system.A, the scheme of the dual-stop mRNA used for quantification of the level of the stop codon readthrough. The mRNA contains an ORF encoding the MVHL tetrapeptide and ending with the first stop codon (UAA, UAG, or UGA). The ORF is followed by a 3՛ context (6 nt) followed by the second stop codon (UAA). When readthrough on the first stop codon occurs, MVHLXXX heptapeptide is synthetized. Created with BioRender.com. B, the scheme of the toe-printing assay used to detect ribosome position on the mRNA. AMV reverse transcriptase synthesizes cDNA on the mRNA template before colliding with the ribosome. The length of cDNA corresponds to position of the ribosome. Created with BioRender.com. C, toe-printing analysis of the ribosomal complexes assembled at the dual-stop mRNAs containing UGA Weak1 context. Different a.a. tRNAs (V, H, L) were added to the initiation complex. An addition of the rabbit tRNA leads to readthrough of the first stop codon and appearance of the preTC2. IC, initiation complex; preTC1 and preTC2, pretermination complexes at the first and second stop codons. a.a. aminoacylated; cDNA, complementary DNA; preTC, pretermination complex.