The Mechanism of tRNA splicing in trypanosomes

锥虫中 tRNA 剪接的机制

基本信息

批准号：
9531616
负责人：
Juan D Alfonzo
金额：
$ 43.89万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9531616
关键词：
Active Sites Alleles Amino Acids Anticodon Area Biochemical Biology Capsicum Chemicals Codon Nucleotides Complex Cyclic Nucleotides Data Defect Disease Drug Targeting Ensure Enzymes Eukaryota Event Evolution Excision Exons Exonuclease Future Genetic Transcription Human In Vitro Interruption Introns Knowledge Lead Left Leishmania Ligase Ligation Link Mammals Medical Messenger RNA Modeling Modification Mutation Nature Nerve Degeneration Neurobiology Nucleotides Organism Outcome Parasites Pathway interactions Phenotype Phosphotransferases Physiological Positioning Attribute Process Protein Biosynthesis Proteins Publishing RNA RNA Editing RNA Interference RNA Processing RNA Splicing Reaction Recombinants Research Role Series Specificity Structure Syndrome System Tail Testing Time Transfer RNA Trypanosoma Trypanosoma brucei brucei Tyrosine Tyrosine-Specific tRNA defined contribution endonuclease enzyme reconstitution in vivo inorganic phosphate insight member mutant phosphoric diester hydrolase tool

项目摘要

In all organisms, tRNAs are matured by a series of post-transcriptional processing events before they can partake in protein synthesis. These include end trimming to generate the correct 5' and 3' ends, chemical modification to ensure proper structure and function, and addition of a 3' CCA tail where amino acids are ultimately attached. In addition a variable number of tRNAs also contain introns; these interrupt the anticodon sequence and as such render a tRNA non-functional. Therefore, splicing is essential for viability. Splicing of tRNA introns differs from mRNA splicing in that it involves several protein-catalyzed steps, including intron cleavage by a splicing-specific endonuclease, followed by exon-end maturation and ligation by a tri-functional ligase (a fusion of three different enzyme domains, depending on the organism) and lastly removal of a “dangling” phosphate left behind by previous activities. In trypanosomatids, there is a single intron-containing tRNA, tRNATyr, responsible for decoding all the tyrosine codons. To date little is known about the mechanism(s) of tRNA splicing in any early diverging protist. Our studies have have identified putative components for each step of the tRNA splicing reaction, each with unusual features. The intron is edited at several positions and editing is in turn required for splicing. The splicing endonuclease has a sub-unit structure where one of the critical sub-units in other systems is missing. The splicing ligase forms a higher order complex suggesting its association with additional factors. The phosphotransferase has an unusual exonuclease domain suggesting an additional role in intron degradation. This proposal will focus in the characterization of each component of the tRNA splicing pathway in T. brucei and will also explore the contribution of intron editing to splicing specificity. Given that the T. brucei mechanism is peppered with unique features, successful completion of these studies will generate important basic information on the mechanism and evolution of tRNA splicing, a poorly understood area of protist biology. !

在所有生物体中，TRNA都通过一系列转录后处理成熟事件在它们可以参与蛋白质合成之前。这些包括修剪到生成正确的5'和3'末端，化学修饰以确保正确的结构和功能，以及最终附着氨基酸的3'CCA尾巴。在成瘾可变数量的TRNA还包含内含子；这些打断了反密码子序列以及这样的tRNA非功能。因此，剪接对于生存能力。 tRNA inron的剪接与mRNA剪接不同，因为它涉及几个蛋白质催化的步骤，包括剪接特异性内切酶的内含子裂解，然后通过三功能连接酶进行外显端的成熟和连接（融合三个不同的酶结构域，具体取决于生物体），最后去除先前活动留下的“悬空”磷酸盐。在锥虫中，有一个含含有器的tRNA，trnatyr，负责解码所有酪氨酸密码子。迄今为止，关于任何早期不同的trNA剪接机制知之甚少原生物。我们的研究已经确定了tRNA每个步骤的假定组件剪接反应，每个反应具有异常特征。内含子在多个位置进行了编辑剪辑又需要编辑。剪接内切核酸酶有一个子单位在其他系统中缺少关键子单元之一的结构。剪接连接酶形成一个高阶复合物，表明其与其他因素相关。磷酸转移酶具有不寻常的核酸外切酶结构域，提示内含子降解中的作用。该提议将集中于每个建议的表征 T. Brucei中tRNA剪接途径的组成部分，也将探索内含子编辑对剪接特异性的贡献。鉴于布鲁氏菌机制是这些研究的成功完成将产生独特的功能，关于tRNA剪接的机制和演变的重要基本信息，差的了解原生物学的领域。呢