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 in vivo system where the reporter protein is secreted from the cells in culture thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media. Using this system, cell cultures 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-course experiments. In particular with this system it is possible to assess very low levels of stop codon suppression. The reporter enzyme, secreted alkaline phosphatase (SEAP), becomes tagged with the S-peptide when there is readthrough 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 activity 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, where a small but significant level of readthrough (suppression) could be detected. We have also characterized suppression of the three stop codons individually, and especially UGA is prone for wobbling.
Sogaard
T M
TM
Institute of Molecular and Structural Biology, University of Aarhus, Aarhus C, DK-8000, Denmark.
Jakobsen
C G
CG
Justesen
J
J
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D018388:Codon, Terminator / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D015870:Gene Expression; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D010303:Paromomycin / Q000494:pharmacology; D010443:Peptide Chain Termination, Translational / Q000187:drug effects; D010454:Peptide Termination Factors / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D014162:Transfection
D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D011993:Recombinant Fusion Proteins; C087971:peptide-chain-release factor 3; D000431:Ethanol; D010303:Paromomycin; D012343:RNA, Transfer; D000469:Alkaline Phosphatase