Structure and Function of the Elongator Complex

伸长复合体的结构和功能

• 批准号: 8698011
• 负责人: Raven H Huang
• 金额: $ 31.34万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698011
• 关键词: Amyotrophic Lateral Sclerosis; Animal Model; Archaea; Bacteria; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Process; Catalytic Domain; Cell Cycle Regulation; Cell physiology; Cells; Chemicals; Codon Nucleotides; Complex; Crystallization; DNA Damage; Data; Data Set; Defect; Development; Disease; Enzymes; Evolution; Exocytosis; Familial Dysautonomia; Genes; Genetic; Genome; Goals; Growth; Histone Acetylation; Holoenzymes; Homologous Gene; Human; In Vitro; Iron; Laboratories; Lead; Light; Link; Literature; Macromolecular Complexes; Modification; Molecular; Mutation; Neurons; Organism; Phase; Play; Property; Proteins; RNA; RNA Polymerase II; Reaction; Recombinants; Recruitment Activity; Regulation; Reporting; Research; Resolution; Rolandic Epilepsy; Role; Series; Staging; Structure; Study models; Sulfur; Transcriptional Activation; Transfer RNA; Translations; Virus; X-Ray Crystallography; Yeasts; base; cell motility; demethylation; experience; in vivo; insight; molecular recognition; motor neuron degeneration; mutant; nervous system disorder; protein complex; public health relevance; reconstitution; research study; response; success

DESCRIPTION (provided by applicant): Approximately 25% of cytoplasmic tRNAs in eukaryotic organisms experience U34 modification at C5, and the modification is thought to be unique to the eukaryotic organisms. Lack of tRNA U34 modification has a disproportionately detrimental effect on translation of several important proteins, due to the encoding genes of which use biased codons that require the modified U34 in tRNAs for efficient translation. Genetic studies indicated that the Elongator complex is responsible for the early stages of the modification. The Elongator complex was initially isolated as part of hyperphosphorylated RNA polymerase II holoenzyme. It has been shown to be involved in a variety of different cellular activities. Defects in the Elongator complex have been linked to several neurological diseases, such as familial dysautonomia (FD), rolandic epilepsy (RE), and amyotrophic lateral sclerosis (ALS). Although the Elongator complex also has been shown to possess histone acetylation activity, accumulating evidence in the literature indicates that tRNA U34 modification at C5 plays a major role in its cellular functions. The Elongator complex is a large macromolecular complex consisting of six subunits of the Elongator proteins, Elp1-6. The third subunit Elp3 is generally regarded as the catalytic subunit, but the mechanism how the Elongator complex carries out tRNA U34 modification at C5 is unknown. Through bioinformatic analysis, we found that the catalytic subunit Elp3, but not other subunits of the eukaryotic Elongator complex, is present in most archaea, a small number of bacteria, and two viruses. Based on this and other bioinformatic analyses, we propose that, unlike tRNA U34 modification in eukaryotic organisms that requires all six subunits of the Elongator complex, Elp3 alone is sufficient for the same modification reaction in archaea and bacteria. Therefore, archaeal or bacterial Elp3 provides us a simplified platform for the study of the mechanism of tRNA U34 modification at C5. In this application, we will utilize expertise from four laboratories to carry out research with three specific aims. First, we will provide the evidence that Elp3 is involved in tRNA U34 modification at C5 in archaea and bacteria by characterizing the modified U34 in tRNAs isolated from these species. Second, we will perform in vitro reconstitution of tRNA U34 modification at C5 using the recombinant archaeal Elp3 and the Elongator complex isolated from yeast cells. Third, we will carry out structural studies of Elp3 alone as well as in complex with tRNA. The long-term goal of this project is to elucidate the structure and function of the eukaryotic Elongator complex, which, while maintaining the main and evolutionarily ancient function of tRNA U34 modification at C5, may have acquired additional biochemical functions over the course of evolution by recruiting Elp1-2 and Elp4-6.

描述（由申请人提供）：真核生物中大约25%的细胞质trna在C5处经历U34修饰，并且这种修饰被认为是真核生物所特有的。缺乏tRNA U34修饰会对几种重要蛋白质的翻译产生不成比例的不利影响，因为编码基因使用偏倚密码子，需要tRNA中修饰的U34才能有效翻译。遗传研究表明，拉长复合体负责早期阶段的修饰。拉长复合体最初是作为高磷酸化RNA聚合酶II全酶的一部分分离出来的。它已被证明参与多种不同的细胞活动。伸长复合体的缺陷与一些神经系统疾病有关，如家族性自主神经障碍（FD）、罗兰癫痫（RE）和肌萎缩侧索硬化症（ALS）。虽然拉长复合体也被证明具有组蛋白乙酰化活性，但越来越多的文献证据表明，tRNA U34在C5的修饰在其细胞功能中起主要作用。伸长复合物是由伸长蛋白Elp1-6的6个亚基组成的大分子复合物。第三个亚基Elp3通常被认为是催化亚基，但细长复合物如何在C5位点进行tRNA U34修饰的机制尚不清楚。通过生物信息学分析，我们发现催化亚基Elp3存在于大多数古细菌、少数细菌和两种病毒中，而非真核延长体复合体的其他亚基。基于此和其他生物信息学分析，我们提出，与tRNA U34在真核生物中的修饰需要所有6个伸长复合物亚基不同，Elp3单独在古细菌和细菌中就足以进行相同的修饰反应。因此，古细菌或细菌Elp3为我们研究tRNA U34在C5位点的修饰机制提供了一个简化的平台。在这个应用程序中，我们将利用来自四个实验室的专业知识来开展有三个具体目标的研究。首先，我们将通过表征从古细菌和细菌中分离的tRNA中修饰的U34来提供Elp3参与tRNA U34在C5位点修饰的证据。其次，我们将利用重组古细菌Elp3和从酵母细胞中分离的伸长复合物在体外重建tRNA U34在C5位点的修饰。第三，我们将对Elp3进行单独以及与tRNA复合的结构研究。该项目的长期目标是阐明真核生物伸长体复合体的结构和功能，