Functional analysis of the tRNA binding protein Kti12

通过在tRNA修饰中操作，Elongator复合物（Elp 1-Elp 6）确保翻译期间准确的mRNA解码。为了正确表达遗传信息，我们可以期望延伸器永久地活跃。然而，包括我们自己在内的几个研究小组的证据表明，tRNA修饰确实会发生变化，并且存在辅助的Elongator伴侣蛋白（Kti 11-Kti 14）。这表明修饰复合物的活性是受调节的，而不是组成性的。根据这一概念，Elongator的最大亚基（Elp 1）通过激酶（Hrr 25/Kti 14）和磷酸酶（Sit 4）进行动态去磷酸化/磷酸化。此外，通过DFG的支持，我们已经表明，Elongator伴侣蛋白Kti 12显然需要促进激酶Hrr 25的募集，以激活Elongator复合物的磷酸化。在DFG资助的最近工作中，我们通过结晶以及深入的遗传和生化分析发现，Kti 12是tRNA激酶（PSTK）的结构模拟物，优先水解ATP的NTP，并以低于Elongator的亲和力结合tRNA。Kti 12如何精确地与Elongator或Hrr 25相互作用，以及其结合tRNA或水解ATP的能力是否会促进Elongator复合物的tRNA修饰活性，是我们在本项目提案中旨在通过以下方式解决的关键方面：- 使用结构指导的Kti 12诱变产生具有tRNA结合缺陷的kti 12突变体，分析Kti 12的tRNA结合对于Elongator的tRNA修饰所起的作用-使用野生型Kti 12分析Kti 12的ATP酶活性对于Elongator功能所起的作用-- 在使用核苷酸或不可水解类似物的Elongator和tRNA相互作用测定中分析Kti 12型和ATP酶突变体-使用Elongator相互作用分析作为蛋白质网络一部分的Kti 12的Elongator激活剂功能，用野生型Kti 12或tRNA结合和ATP酶突变体的磷酸化和tRNA修饰测定-在从野生型细胞和具有tRNA结合或ATP酶缺陷的突变体中纯化无延伸子的Kti 12库之后，我们的计划将使用体内和体外的方法来鉴定Kti 12的结构-功能要求，以增强我们对tRNA修饰复合物Elongator的调控的理解。随着越来越多的证据表明，Elongator连接的tRNA修饰缺陷与人类的错误翻译，蛋白毒性和神经病变的形成有关，我们的项目具有生物医学意义。因此，更好地了解不适当的tRNA修饰在延长肌功能障碍的条件下所起的作用，可以深入了解疾病综合征的机制，并为治疗干预方案提供信息。

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.