Functional analysis of the tRNA binding protein Kti12

通过作用于tRNA修饰，Elongator复合体(Elp1-Elp6)确保了翻译过程中准确的mRNA解码。因此，为了正确表达遗传信息，人们可能会认为Elongator是永久活跃的。然而，来自包括我们自己在内的几个研究团队的证据表明，tRNA修饰确实会改变，并且存在辅助伸长蛋白伙伴蛋白(Kti11-Kti14)。这表明修饰剂复合体的活性是受调控的，而不是结构性的。与这一概念一致，Elongator最大的亚基(Elp1)经历了由一个激酶(Hrr25/Kti14)和一个磷酸酶(Sit4)的动态去磷酸化。此外，我们通过DFG的支持表明，Elongator伙伴蛋白Kti12显然是促进激活Elongator复合体的磷酸化的激酶Hrr25的募集所必需的。在DFG资助的最近的工作中，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，优先于ATP水解NTPs，并以比Elongator更低的亲和力结合tRNA。Kti12与Elongator或Hrr25相互作用的精确程度以及其结合tRNA或水解ATP的能力是否促进Elongator复合体的tRNA修饰活性是我们在本项目建议书中旨在通过以下方式解决的关键方面：-分析由Kti12结合的tRNA对Elongator修饰tRNA的作用使用结构引导的KTI12突变产生具有tRNA结合缺陷的kti12突变体-使用Elongator中的野生型Kti12和ATPase突变分析Elongator中的ATPase活性对Elongator功能的作用-使用Elongator相互作用分析Kti12作为蛋白质网络的一部分的Elongator激活子功能，野生型Kti12或tRNA结合和ATPase突变的磷酸化和tRNA修饰分析-在从野生型细胞和tRNA结合或ATPase缺失的突变体中纯化无Elongator的Kti12池后，用MALDI-TOF-MS/MS鉴定Elongator无关的Kti12交互作用。综上所述，我们拟议的项目将使用旨在确定Kti12结构-功能要求的体内和体外方法，以加深我们对tRNA修饰复合体Elongator的调控的理解。随着越来越多的证据表明Elongator与tRNA修饰缺陷有关，与人类的误翻译、蛋白毒性和神经疾病的形成有关，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修饰在伸长器功能障碍条件下所起的作用，可能为疾病综合征的机制提供洞察力，并为治疗干预方案提供参考。

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.