Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts.

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.