Structural basis of canonical and non-canonical translation termination and recycling by eRF1/eRF3 and ABCE1 in yeast and humans

项目P3(Beckmann)解决了使用酵母(酿酒酵母)和人类系统进行真核细胞翻译、终止和核糖体回收的基本阶段。在第一个资助期，利用小麦胚芽体外翻译系统产生了功能终止复合体(包含eRF1和eRF3)和预循环复合体(包含eRF1和ABCE1)。为了定位核糖体A位点上的终止密码子，使用了带有停滞序列的病毒多肽。用纯化的终止/回收因子在体外重组这种停滞的核糖体，并以8-9ä分辨率解析组装的复合体的冷冻-EM结构。我们观察到，在对含有eRF3的前终止复合体中的终止密码子进行解码后，eRF1的中央结构域移动到大的60S亚基的肽基转移酶中心，从而释放出含有ABCE1的复合体中的新生多肽。这解释了eRF1在结构水平上如何在翻译终止和核糖体循环中发挥作用。为了获得与人类系统相似的结构，我们建立了可靠、高效的人类体外翻译系统。此外，我们使用不依赖于起始因子的CRPV-IRES优化了产生停滞核糖体的构建体的表达。在此基础上，终止和预循环复合体可以在体外重组，从而产生第一个有希望的冷冻-EM重建。在第二个资助期，我们将继续通过高分辨率冷冻EM(使用新安装的FEI Falcon 2直接电子探测器技术)阐明人类终止和再循环的分子基础，重点是eRF1对终止密码子的识别和多肽-tRNA的水解。

Project P3 (Beckmann) addresses the essential phases of eukaryotic translation termination and ribosome recycling using the yeast (S. cerevisiae) and the human system. In the first funding period functional termination complexes (containing eRF1 and eRF3) and pre-recycling complexes (containing eRF1 and ABCE1) were generated using a wheat germ in vitro translation system. To position a stop-codon in the ribosomal A-site a viral polypeptide with stalling sequence was used. Such stalled ribosomes were in vitro reconstituted with purified termination/recycling factors and cryo-EM structures of assembled complexes were solved at 8-9 Å resolution. We observe that after decoding the stop-codon in the eRF3-containing pre-termination complex the eRF1 central domain moves into the peptidyl-transferase center of the large 60S subunit to release the nascent peptide in the ABCE1-containing complex. This explains how eRF1 functions in both translation termination and ribosome recycling on a structural level. To obtain similar structures in the human system we established a reliable and highly efficient human in vitro translation system. Furthermore we optimized the expression for constructs yielding stalled ribosomes by using the initiation factor independent CrPV-IRES. On this basis termination and pre-recycling complexes could be reconstituted in vitro resulting in first promising cryo-EM reconstructions. In the second funding period we will continue to elucidate the molecular basis of human termination and recycling by high-resolution cryo-EM (using newly installed FEI Falcon 2 direct electron detector technology) with a focus on stop-codon recognition and peptidyl-tRNA hydrolysis by eRF1.