Kandala Divya T, Del Piano Alessia, Minati Luca, Clamer Massimiliano
IMMAGINA BioTechnology S.r.l, Via alla cascata 56/c, Povo, Trento 38123, Italy.
ACS Omega. 2019 Jun 13;4(6):10336-10345. doi: 10.1021/acsomega.9b00366. eCollection 2019 Jun 30.
Puromycin is a well-known antibiotic that is used to study the mechanism of protein synthesis and to monitor ribosome activity due to its incorporation into nascent peptide chains. However, puromycin effects outside the ribosome catalytic core remain unexplored. Here, we developed two analogues (3PB and 3PC) of the 3'-end of tyrosylated-tRNA that can efficiently interact with several proteins associated with ribosomes. We biochemically characterized the binding of these analogues and globally mapped the direct small molecule-protein interactions in living cells using clickable and photoreactive puromycin-like probes in combination with in-depth mass spectrometry. We identified a list of proteins targeted by the molecules during ribosome activity (e.g., GRP78), and we addressed possible uses of the probes to sense the activity of protein synthesis and to capture associated RNA. By coupling genome-wide RNA sequencing methods with these molecules, the characterization of unexplored translational control mechanisms will be feasible.
嘌呤霉素是一种著名的抗生素，由于其可掺入新生肽链中，因此被用于研究蛋白质合成机制并监测核糖体活性。然而，嘌呤霉素在核糖体催化核心之外的作用仍未得到探索。在此，我们开发了两种酪氨酰化tRNA 3'末端的类似物（3PB和3PC），它们能够有效地与几种核糖体相关蛋白相互作用。我们对这些类似物的结合进行了生化表征，并结合深度质谱，使用可点击和光反应性嘌呤霉素样探针在活细胞中全面绘制了直接的小分子-蛋白质相互作用图谱。我们确定了分子在核糖体活性过程中靶向的一系列蛋白质（例如，GRP78），并探讨了这些探针在检测蛋白质合成活性和捕获相关RNA方面的可能用途。通过将全基因组RNA测序方法与这些分子相结合，对未探索的翻译控制机制进行表征将成为可能。
