Raw data from all measurements.

Translational termination is not entirely efficient and competes with elongation, which might result in translational readthrough (TR). TR occurs when a near-cognate tRNA binds to a stop codon, (mis)interpreting it as a sense codon and producing a C-terminal extension of the protein. This process is influenced by the stop codon itself and the surrounding nucleotide sequence, known as the stop codon context (SCC). To investigate the role of these cis-acting elements beyond the high-TR motif UGA CUA G, this study examines specific positions within the SCC, both upstream and downstream of the motif, that contribute to variations in basal and aminoglycoside-induced TR. In particular, we identified a surprisingly large influence of the upstream nucleotide positions -9 and -8 (relative to the stop codon) and positions +11 and +12 on readthrough levels, revealing a complex interplay between nucleotides in the expanded SCC with effects turning out to be non-linear and, furthermore, not transferable to evolutionarily non-adapted SCCs. These findings support our understanding of translational termination and may benefit the development of pharmacological therapy for diseases caused by premature stop codon mutations.