Correlating dynamics with function in the eukaryotic exoribonuclease enzymes Xrn1 and Xrn2

将动力学与真核核糖核酸外切酶 Xrn1 和 Xrn2 的功能相关联

• 批准号: 501637118
• 负责人: Professor Dr. Remco Sprangers
• 金额: --
• 依托单位: Lehrstuhl für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/501637118?language=en
• 关键词: Correlating; dynamics; function; eukaryotic; exoribonuclease

Eukaryotic 5′→3′ exoribonucleases are large (> 100 kDa) enzymes that remove nucleotides from the 5’ end of single stranded RNA in a processive manner, thereby releasing mononucleotides. Eukaryotic organisms contain two closely related 5′→3′ exoribonucleases: the cytoplasmic Xrn1 enzyme (also called PACMAN) that is involved in the degradation of mRNA and the nuclear Xrn2 enzyme (also called Rat1) that plays a role in the degradation and processing of a variety of RNA species. The few known static structures of Xrn1 and Xrn2 reveal that the active site is only accessible for single-stranded RNA. Differences between the determined structures, however, suggest that the processive translocation of the substrate towards the active site is associated with conformational changes in the enzymes. The aim of this proposal is to address how these and other dynamics processes in Xrn1 and Xrn2 correlate with function. Experimentally, we will make extensive use of methyl TROSY and 19F NMR spectroscopic methods to directly quantify motions in Xrn1 and Xrn2 with a high spatial resolution. In preliminary work, we have shown that these approaches yield high quality data that can provide detailed insights into populations and exchange rates of the associated conformational states. Our extensive NMR studies will thus provide an extensive and unique picture of the conformantional dynamics of these enzymes. Changes of dynamics in the presence of inhibitors, substrates and binding partners (e.g. DcpS and Rai1 that modulate the activity of Xrn1, respectively Xrn2) or in the absence of specific domains will enable insights into the enzymatic mechanisms that regulate catalytic activity. In addition, we will design Xrn1 and Xrn2 enzymes that display altered dynamics and determine how this influences the catalytic turnover rates. Combined, these data will allow us to establish a (potentially) direct functional link between enzyme dynamics and catalytic activity. In brief, our proposed work goes well beyond the static structural information that is available for Xrn1 and Xrn2. In particular, we will gain unique insights into dynamic processes that are important for catalytic activity and the regulation thereof. These findings will have general implications for our understanding of enzymes, as data that link motions with function are sparse. In addition, our NMR work is at the forefront of what is currently technically possible.

真核生物的5 ‘→3 ’外核糖核酸酶是一种大的（> 100 kDa）酶，它以一种过程的方式从单链RNA的5 '端去除核苷酸，从而释放单核苷酸。真核生物含有两种密切相关的5 ‘→3 ’外核糖核酸酶：细胞质Xrn1酶（也称为PACMAN）参与mRNA的降解，核Xrn2酶（也称为Rat1）参与多种RNA的降解和加工。少数已知的Xrn1和Xrn2的静态结构表明，只有单链RNA才能进入活性位点。然而，确定的结构之间的差异表明，底物向活性位点的过程易位与酶的构象变化有关。本提案的目的是解决Xrn1和Xrn2中的这些和其他动态过程如何与功能相关。实验上，我们将广泛使用甲基TROSY和19F核磁共振光谱方法，以高空间分辨率直接量化Xrn1和Xrn2中的运动。在初步工作中，我们已经证明这些方法产生了高质量的数据，可以提供有关相关构象态的种群和交换率的详细见解。因此，我们广泛的核磁共振研究将为这些酶的构象动力学提供广泛而独特的图像。在抑制剂、底物和结合伙伴（例如分别调节Xrn1和Xrn2活性的dcp和Rai1）存在或缺乏特定结构域的情况下，动力学的变化将使我们能够深入了解调节催化活性的酶促机制。此外，我们将设计显示改变动力学的Xrn1和Xrn2酶，并确定这如何影响催化周转率。综合起来，这些数据将使我们能够在酶动力学和催化活性之间建立（潜在的）直接功能联系。简而言之，我们建议的工作远远超出了Xrn1和Xrn2可用的静态结构信息。特别是，我们将获得对催化活性及其调控重要的动态过程的独特见解。这些发现将对我们对酶的理解产生普遍影响，因为将运动与功能联系起来的数据很少。此外，我们的核磁共振工作是在什么是目前技术上可行的最前沿。