Figure 3. 8-oxoG stalls translation in cell extracts.

(A) Schematic of the mRNA reporters used in eukaryotic extracts. The full-length mRNA encodes a peptide that has a molecular weight of ~9 kDa, whereas the stop mRNA, which has a stop codon at the position of the 8-oxoG codon in the 8-oxoG mRNA, encodes a peptide that has a molecular weight of ~8.4 kDa. Reporters used in S30 reactions were identical minus the cap and polyadenylation. (B) Autoradiograph of a bis-Tris gel of translation assays using bacterial S30 extracts. The 8-oxoG transcript yields a peptide of a size similar to the stop reporter. * indicates non-specific band. (C) Autoradiograph of bis-Tris and Tris-Tricine gels of in vitro translation assays in wheat germ extracts. Proteins were labeled by the addition of [³⁵S]-Methionine to the reactions. The 8-oxoG mRNA and no-stop mRNA both accumulate peptidyl-tRNA, visible on the bis-Tris gel, which disappear upon the addition of RNase A. Reactions separated on Tris-Tricine were incubated with and without MG132. The 8-oxoG mRNA produces truncated protein products (marked by arrows); the largest of these has a size similar to that observed in the presence of stop mRNA. See also Figure S3.