Department of Biology, Molecular Microbiology, University of Konstanz, 78457, Konstanz, Germany.
Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, 78457, Konstanz, Germany.
Sci Rep. 2021 Apr 21;11(1):8681. doi: 10.1038/s41598-021-87847-y.
The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.
真核生物核糖体相关复合物（RAC）在从头蛋白质折叠中发挥重要作用。它与核糖体的独特相互作用，包括与两个核糖体亚基的接触，表明 RAC 介导的翻译延伸和共翻译蛋白质折叠之间的协调作用。在这里，我们应用电子顺磁共振（EPR）光谱结合定点自旋标记（SDSL）技术，深入了解影响翻译准确性的 RAC-核糖体接触点。我们确定了 RAC 与核糖体的局部接触点。这些数据首次提供了全长 RAC 在溶液中和核糖体上存在四螺旋束和长α螺旋的实验证据。此外，我们还补充了介导该功能重要接触的区域在 40S 核糖体亚基上的结构图像。总之，本研究首次应用 SDSL-EPR 光谱学阐明了 3.3 MDa 翻译机制与伴侣复合物之间相互作用的分子细节。
