Figure 3.

The ORF2 AUG is dispensable for retrotransposition. (A) Mutating the AUG codon to either AUA or CCC is still compatible with retrotransposition. The schematic depicts cartoons of each of the mutant constructs tested in the retrotransposition assay. The respective mutations (AUG to AUA in RA102; AUG to CCC in RA103; AUGACAGGA to CCCUAAUAA in RAPXX) are indicated below the cartoon. Construct names are indicated in the left column, and representative data from the cultured cell retrotransposition assay are indicated at the right of the figure. The relative retrotransposition efficiency is indicated and is expressed as compared with the relative retrotransposition efficiency of the wild-type control (RA101). (B) Exogenous sources of RT are not promoting retrotransposition of the mutant constructs. Double mutants containing either the M1P or M1X mutation in conjunction with an RT active site mutation (RA103/RT- or RA111/RT-) were assayed for retrotransposition. Both mutants were retrotransposition defective, indicating that ORF2p was translated in the original M1P and M1X mutants. (C) The ORF2 AUG codon can be substituted with any coding triplet. The ORF2 AUG was mutated individually so that it could encode the other 19 amino acids. Each of the resultant constructs (X-axis) retrotransposed at 10%-70% of wild-type levels (Y-axis). By comparison, mutating the AUG to each stop codon (Opal, Ochre, and Amber) reduced retrotransposition by ∼50-fold. The percent of retrotransposition is shown compared with the wild-type element RA101 (i.e., M1M in the bar graph). The error bars indicate the standard deviation, which was calculated from at least six independent experiments for each construct.