A sensitive assay of translational fidelity (readthrough and termination) in eukaryotic cells

The process of translation termination in eukaryotes has been monitored by different types of assays, each with its own merits. We have developed an i n vivo system where the reporter protein is secreted from the cells in cult ure thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media. Using this system, cell cult ures can be challenged with various stimuli during growth and the cellular responses on the translational level can be investigated in vivo as well as in vitro. Sampling is rapid, easy, and non-destructive to the cells, which enables measurement of translational fidelity in real time during time-cou rse experiments. In particular with this system it is possible to assess ve ry low levels of stop codon suppression. The reporter enzyme, secreted alka line phosphatase (SEAP), becomes tagged with the S-peptide when there is re adthrough of a stop codon placed between the C-terminus of the SEAP and the S-peptide. The tagged SEAP is bound to a matrix and the bound SEAP activit y is measured versus total SEAP activity in the medium as a reference. With this assay we have confirmed that eRF1 acts as an antisuppressor in cells transfected with a cognate suppressor tRNA as well as in control cells, whe re a small but significant level of readthrough (suppression) could be dete cted. We have also characterized suppression of the three stop codons indiv idually, and especially UGA is prone for wobbling.