Dynamics and mechanisms of Argonaute proteins and the RISC complex studied with single molecule fluorescence spectroscopy

来自生命所有三个领域的Argonaute （AGO）蛋白在特异性调节细胞核酸水平的过程中起关键作用。其中一些AGO蛋白，包括人类Argonaute2 （hAGO2）和来自古生菌jannaschii甲烷钙球菌（MjAgo）的AGO，能够切割通过与AGO相关的短互补引导链识别的核酸靶链。人类AGO2和MjAGO是真核生物和原核生物AGO分支的代表，它们共享一个保守的四域结构。然而，eAGOs和pAGOS的生物学作用有显著差异。作为真核RNA干扰通路的关键组分，hAGO2参与了高达60%的基因转录后调控。在这个过程中失败可能会带来毁灭性的后果，从糖尿病和几种癌症到心力衰竭。相比之下，原核AGO蛋白参与细胞防御针对DNA而非RNA的外来核酸，并能够以导向依赖和导向独立的方式运作。然而，对这一过程及其调控的了解仍然很少。我们已经开发了生物化学，结构和尖端的光学单分子方法，使我们能够阐明原核和真核AGO蛋白的结构-功能-动力学关系。通过我们的研究，我们打算加深对eAGO和pAGO机制的理解，以及AGO蛋白在其整个活性周期中所采样的构象空间，从而阐明这些结构保守的蛋白如何能够在生物高度多样化的途径中发挥作用。我们将重点关注以下问题：我们实验室对MjAGO的晶体结构和功能研究表明，MjAGO具有一个由底物亚群占据的二级核酸结合通道。我们将描述结合通道占用和伴随的构象变化。此外，我们的目标是阐明复杂的底物（如质粒DNA）如何在原核生物的AGOs中被容纳，以及哪些调节因子控制MjAGO的作用。hAGO2的活性受到多种蛋白和核酸相互作用伙伴的协调，并受到翻译后修饰的调节。这些相互作用物和修饰如何影响hAGO2的结构组织尚不清楚。我们的目标是捕获含hago2配合物的不同阶段，以确定这些配合物的结构组织和动态采用单分子FRET测量。另一个重点将是分析人类AGO蛋白与预处理核酸酶Dicer在单分子水平上的相互作用。

Argonaute (AGO) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these AGO proteins, among them human Argonaute2 (hAGO2) and AGO from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an AGO-associated short complementary guide strand. Human AGO2 and MjAGO are representatives of the eukaryotic and prokaryotic AGO clade that share a conserved four-domain architecture. However, the biological roles of eAGOs and pAGOS differ significantly. As key components of the eukaryotic RNA interference pathway, hAGO2 takes part in the finely tuned post-transcriptional regulation of up to 60% of our genes. Failure in this process can have devastating consequences ranging from diabetes and several forms of cancer to heart failure. In contrast, prokaryotic AGO proteins are implicated to partake in the cellular defence against foreign nucleic acids targeting DNA rather than RNA and are able to operate in a guide-dependent and guide-independent manner. The understanding of this process and its regulation, however, remains sparse. We have developed biochemical, structural and cutting-edge optical single-molecule methods that allow us to illuminate the structure-function-dynamics relationship of prokaryotic and eukaryotic AGO proteins. With our investigations we intend to deepen the understanding of eAGO and pAGO mechanisms and the conformational space sampled by AGO proteins throughout their activity cycle thereby shedding light on the question how these structurally conserved proteins are able to function in biologically highly diverse pathways. We will focus on the following questions:Crystal structures and functional studies of MjAGO from our laboratory indicate that MjAGO possesses a secondary nucleic acid binding channel that is occupied by a subset of substrates. We will characterise the binding channel occupancy and accompanying conformational changes. Furthermore, we aim to elucidate how complex substrates (e.g. plasmid DNA) are accommodated in prokaryotic AGOs and which regulating factors control the action of MjAGO. The activity of hAGO2 is orchestrated by various protein and nucleic acid interaction partners and modulated by post-translational modifications. How these interactors and modifications influence the structural organisation of hAGO2 is poorly understood. We aim to capture different stages of hAGO2-containing complexes to determine the structural organisation and dynamics of these complexes employing single-molecule FRET measurements. An additional major focus will be the analysis of the interaction between human AGO proteins with the pre-processor nuclease Dicer on the single-molecule level.