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Structure and Function of the Elongator Complex

伸长复合体的结构和功能

基本信息

批准号：
9325029
负责人：
Raven H Huang
金额：
$ 28.82万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9325029
关键词：
Amyotrophic Lateral Sclerosis Anaerobic Bacteria 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 study 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 Structure Study models Sulfur Transcriptional Activation Transfer RNA Translations Virus X-Ray Crystallography Yeasts base cell motility demethylation experience experimental study in vivo insight molecular recognition motor neuron degeneration mutant nervous system disorder protein complex public health relevance reconstitution response success

项目摘要

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）：真核生物中约25%的细胞质tRNA在C5处经历U34修饰，并且认为该修饰是真核生物所特有的。缺乏tRNA U34修饰对几种重要蛋白质的翻译具有不成比例的不利影响，这是由于其编码基因使用偏向密码子，需要tRNA中修饰的U34来进行有效翻译。遗传学研究表明，延长复合体负责早期阶段的修改。延伸体复合物最初是作为过度磷酸化的RNA聚合酶II全酶的一部分分离的。它已被证明参与各种不同的细胞活动。延长肌复合体的缺陷与多种神经系统疾病有关，例如家族性自主神经功能障碍（FD）、罗兰癫痫（RE）和肌萎缩侧索硬化症（ALS）。尽管Elongator复合物也被证明具有组蛋白乙酰化活性，但文献中积累的证据表明，C5处的tRNA U34修饰在其细胞功能中起主要作用。Elongator复合物是由Elongator蛋白的六个亚基Elp 1 -6组成的大分子复合物。第三亚基Elp 3通常被认为是催化亚基，但Elongator复合物如何在C5处进行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Elp 3，而不是真核拉长复合物的其他亚基，存在于大多数古细菌，少数细菌，和两种病毒。基于这一点和其他生物信息学分析，我们提出，不像tRNA U34修饰在真核生物中，需要所有六个亚基的延伸复合物，Elp 3单独是足够的古细菌和细菌中相同的修饰反应。因此，古细菌或细菌Elp 3为我们研究tRNA U34在C5的修饰机制提供了一个简化的平台。在这项申请中，我们将利用四个实验室的专业知识，以三个具体目标进行研究。首先，我们将提供证据表明，Elp 3是在古细菌和细菌中的C5的tRNA U34的修饰，通过表征从这些物种中分离的tRNA的U34的修饰。其次，我们将使用从酵母细胞分离的重组古细菌Elp 3和Elongator复合物在C5处进行tRNA U34修饰的体外重建。第三，我们将进行Elp 3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延长子复合物的结构和功能，虽然保持了C5处tRNA U34修饰的主要和进化上古老的功能，但可能在进化过程中通过募集Elp 1 -2和Elp 4 -6获得了额外的生化功能。