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）：真核生物中大约25％的细胞质TRNA经历了C5时的U34修饰，并且该修饰被认为是真核生物所独有的。缺乏tRNA U34修饰对几种重要蛋白的翻译产生不成比例的有害作用，这是因为编码基因使用偏置的密码子，这些密码子需要在TRNA中进行修改的U34以进行有效翻译。遗传研究表明，伸长膜复合物是修饰的早期阶段。首先将延伸仪复合物分离为热磷酸化RNA聚合酶II全酶的一部分。已显示它与多种不同的细胞活性有关。伸长剂复合物中的缺陷已与几种神经系统疾病有关，例如家族性动物障碍（FD），Rolandic癫痫（RE）和肌萎缩性侧面硬化症（ALS）。尽管还显示出伸长量复合物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。伸长仪复合物是一个大的大分子复合物，由六个亚基ELP1-6组成。第三个亚基ELP3通常被视为催化亚基，但是延伸仪复合物在C5处进行tRNA U34修饰的机制尚不清楚。通过生物信息学分析，我们发现大多数古细菌，少数细菌和两种病毒都存在催化亚基ELP3，但没有真核延伸器复合物的其他亚基。基于此和其他生物信息学分析，我们提出，与真核生物中的tRNA U34修饰不同，需要延伸剂复合物的所有六个亚基，仅ELP3就足以足以在古细菌和细菌中进行相同的修饰反应。因此，古细菌或细菌ELP3为我们提供了一个简化的平台，用于研究C5时TRNA U34修饰机理。在此应用程序中，我们将利用四个实验室的专业知识来进行三个特定目标的研究。首先，我们将提供证据表明，通过表征与这些物种分离的TRNA中的修饰的U34，ELP3参与了C5和细菌的tRNA U34修饰。其次，我们将使用重组古细胞ELP3和从酵母细胞分离的延伸仪复合物在C5处进行tRNA U34修饰的体外重构。第三，我们将单独进行ELP3以及与tRNA的复杂性进行结构研究。该项目的长期目标是阐明真核伸长膜络合物的结构和功能，该复合物的结构和功能同时，通过募集ELP1-2和ELP4-6，在C5处的tRNA U34修饰的主要古代函数可能在进化过程中获得了其他生化功能。