Analyses of the dynamic interplay between ribosomes and bacterial translocon complexes

膜包埋的转位子复合物与翻译核糖体的相互作用是蛋白质分选的一个重要先决条件，无论是在欧盟和原核生物。虽然核糖体的翻译活性足以将一些细菌膜蛋白整合到脂质相中，但许多其他蛋白还需要ATP酶SecA。SecA如何能够在核糖体保持附着于SecYEG易位子的同时进入新生链目前尚不清楚。我们的目标是确定动态相互作用的核糖体和SecA在积极运输SecYEG translocons通过定点交联和解剖的不同阶段的运输使用重建测定。普遍保守的YidC作为细菌整合位点起作用，既与SecYEG一致，也独立于SecYEG。关于蛋白质如何靶向YidC以及这是否是涉及细菌SRP途径的严格共翻译过程，存在相互矛盾的数据。还未知YidC在共翻译整合期间如何与SecYEG相互作用，以及SecYEG依赖性和非依赖性底物是否被YidC不同地处理。我们希望通过结合体内技术，如定点体内交联和FRET与体外技术，如体外转运试验，使用重组蛋白脂质体和亲和力测量来解决这些问题。

The interaction of membrane-embedded translocon complexes with translating ribosomes is a crucial prerequisite for protein sorting in both eu- and prokaryotic organisms. Although the translational activity of the ribosome is sufficient for integrating some bacterial membrane proteins into the lipid phase, many others require the ATPase SecA in addition. How SecA is able to access a nascent chain while the ribosome stays attached to the SecYEG translocon is currently unknown. Our goal is to determine the dynamic interaction of ribosomes and SecA in actively transporting SecYEG translocons by site-directed cross-linking and by dissecting the different stages of transport using reconstitution assays. The universally conserved YidC functions as a bacterial integration site, both in concert with SecYEG but also independently of SecYEG. There are conflicting data on how proteins are targeted to YidC and whether this is a strict co-translational process involving the bacterial SRP pathway. It is also unknown, how YidC interacts with SecYEG during co-translational integration and whether SecYEGdependent and –independent substrates are handled differently by YidC. We want to address these questions by combining in vivo techniques like site-directed in vivo cross-linking and FRET with in vitro techniques, like in vitro transport assays using reconstituted proteoliposomes and affinity measurements.