Department of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece.
Biochimie. 2013 Sep;95(9):1765-72. doi: 10.1016/j.biochi.2013.06.004. Epub 2013 Jun 14.
The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.
核糖体出口隧道最近成为许多功能和结构研究的中心。越来越多的证据表明，该隧道不仅仅是新生链的简单被动通道，而是一个相当重要的功能区域，在这个区域中，新生肽序列可以与核糖体相互作用，以发出信号使翻译减速甚至停止。为了进一步探索这种相互作用，我们合成了附着在氯霉素（CAM）碱基氨基上的短肽，使得当这些化合物结合到核糖体上时，它们模拟了结合到肽基转移酶中心（PTC）A 位的新生肽酰-tRNA 链。在这里，我们表明这些 CAM-肽与核糖体的 PTC 相互作用，而它们的有效性可以通过肽的序列来调节，这表明肽与核糖体隧道的直接相互作用。事实上，在存在 CAM-P2（测试的 CAM-肽之一）的情况下进行的化学足迹分析揭示了位于隧道深处的 23S rRNA 核苷酸的保护，表明与核糖体隧道的特定成分可能存在相互作用。总的来说，我们的发现表明，基于 CAM 的肽衍生物将成为将多肽链模拟物靶向核糖体隧道的有用工具，允许使用进一步的生化和结构方法来探索它们的构象和与核糖体隧道的相互作用。
