Functional analysis of the tRNA binding protein Kti12

tRNA结合蛋白Kti12的功能分析

• 批准号: 450558823
• 负责人: Professor Dr. Raffael Schaffrath
• 金额: --
• 依托单位: Fachgebiet Mikrobiologie
• 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/450558823?language=en
• 关键词: Functional; analysis; tRNA; binding; protein

By operating in tRNA modification, the Elongator complex (Elp1-Elp6) assures accurate mRNA decoding during translation. For proper expression of genetic information, one may therefore expect Elongator to be permanently active. Evidence from several research teams including our own, however, shows that tRNA modifications do change and that there are accessory Elongator partner proteins (Kti11-Kti14). This suggests the activity of the modifier complex is regulated rather than constitutive. In line with this notion, Elongator’s largest subunit (Elp1) undergoes dynamic de/phosphorylation by a kinase (Hrr25/Kti14) and a phosphatase (Sit4). In addition, we have shown through DFG support that Elongator partner protein Kti12 apparently is required to promote recruitment of kinase Hrr25 for activating phosphorylation of the Elongator complex.In recent work funded by the DFG, we discovered by crystalization as well as in-depth genetic and biochemical analyses that Kti12 is a structural mimic of a tRNA-Kinase (PSTK), hydrolyses NTPs with preference for ATP and binds tRNAs with an affinity lower than Elongator. How precisely Kti12 interacts with Elongator or Hrr25 and whether its capacity to bind tRNA or hydrolyze ATP promotes the tRNA modification activity of the Elongator complex are key aspects we aim to address in this project proposal in the following ways:- analysis of the role tRNA binding by Kti12 plays for tRNA modification by Elongator using structure-guided KTI12 mutagenesis for generation of kti12 mutants with tRNA binding defects- analysis of the role ATPase activity of Kti12 plays for Elongator function using wild-type Kti12 and ATPase mutants in Elongator and tRNA interaction assays with nucleotides or non-hydrolyzable analoga- analysis of the Elongator activator function of Kti12 as part of a protein network using Elongator interaction, phosphorylation and tRNA modification assays with wild-type Kti12 or tRNA binding and ATPase mutants- identification of Elongator unrelated Kti12 interactors by MALDI-TOF-MS/MS after purification of Elongator-free Kti12 pools from wild-type cells and mutants with tRNA binding or ATPase defectsIn sum, our proposed project will use in vivo & in vitro methods aimed at identifying Kti12 structure-function requirements to enhance our understanding about the regulation of the tRNA modifier complex Elongator. With a growing body of evidence showing that Elongator linked tRNA modification defects associate with mistranslation, proteotoxicity and formation of neuropathies in humans, our project has biomedical relevance. Thus, a better knowledge about the roles played by inappropriate tRNA modifications under conditions of Elongator dysfunction may provide insights into disease syndrome mechanisms and inform therapeutic intervention schemes.

通过tRNA修饰，拉长复合体（Elp1-Elp6）在翻译过程中确保准确的mRNA解码。因此，对于遗传信息的适当表达，人们可以期望伸长子永久活跃。然而，包括我们自己的研究小组在内的几个研究小组的证据表明，tRNA修饰确实发生了变化，并且存在辅助的伸长伴侣蛋白（Kti11-Kti14）。这表明修饰语复合物的活性是受调节的，而不是组成的。与这一概念一致，伸长子的最大亚基（Elp1）经历了激酶（Hrr25/Kti14）和磷酸酶（Sit4）的动态去磷酸化。此外，我们已经通过DFG支持表明，延长体伴侣蛋白Kti12显然需要促进激酶Hrr25的募集，以激活延长体复合物的磷酸化。在最近由DFG资助的工作中，我们通过结晶以及深入的遗传和生化分析发现Kti12是tRNA-Kinase （PSTK）的结构模拟物，优先水解ATP的NTPs，并以低于伸长子的亲和力结合trna。如何精确Kti12与延伸机或Hrr25和其能力是否绑定tRNA或水解ATP促进tRNA修改活动延伸机复杂的关键方面我们的目标是解决在这个项目建议在以下方式:-分析Kti12 tRNA绑定角色的扮演tRNA修改通过延伸机使用structure-guided Kti12诱变与tRNA绑定生成Kti12突变体的缺陷——分析Kti12扮演的角色腺苷三磷酸酶活动吗使用野生型Kti12和ATPase突变体进行延长子和tRNA相互作用的核苷酸或不可水解类似物分析-使用延长子相互作用分析Kti12的延长子激活子功能作为蛋白质网络的一部分野生型Kti12或tRNA结合和ATPase突变体的磷酸化和tRNA修饰试验-从野生型细胞和tRNA结合或ATPase缺陷突变体中纯化无伸长体Kti12池后，使用MALDI-TOF-MS/MS鉴定与伸长体无关的Kti12相互作用物。我们提出的项目将使用体内和体外方法，旨在确定Kti12的结构-功能需求，以增强我们对tRNA修饰复合物伸长器调控的理解。随着越来越多的证据表明，细长体相关的tRNA修饰缺陷与误翻译、蛋白质毒性和人类神经病变的形成有关，我们的项目具有生物医学意义。因此，更好地了解在伸长体功能障碍条件下不适当的tRNA修饰所起的作用，可能为疾病综合征机制提供见解，并为治疗干预方案提供信息。