Sogaard T M, Jakobsen C G, Justesen J
Institute of Molecular and Structural Biology, University of Aarhus, Aarhus C, DK-8000, Denmark.
Biochemistry (Mosc). 1999 Dec;64(12):1408-17.
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.
真核生物中翻译终止过程已通过不同类型的检测方法进行监测，每种方法都有其自身的优点。我们开发了一种体内系统，其中报告蛋白从培养的细胞中分泌出来，从而能够通过简单地采集细胞培养基来持续监测翻译终止活性。利用该系统，细胞培养物在生长过程中可以受到各种刺激，并且可以在体内和体外研究翻译水平上的细胞反应。采样快速、简便且对细胞无损伤，这使得在时间进程实验中能够实时测量翻译保真度。特别是使用该系统，可以评估非常低水平的终止密码子抑制。当位于分泌型碱性磷酸酶（SEAP）C末端和S肽之间的终止密码子发生通读时，报告酶分泌型碱性磷酸酶（SEAP）会与S肽标记在一起。标记的SEAP与基质结合，并将结合的SEAP活性与培养基中的总SEAP活性作为参考进行测量。通过该检测方法，我们证实了在转染了同源抑制性tRNA的细胞以及对照细胞中，eRF1作为一种抗抑制因子发挥作用，在对照细胞中可以检测到少量但显著水平的通读（抑制）。我们还分别对三个终止密码子的抑制情况进行了表征，特别是UGA容易发生摆动。
