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 通过一系列转录后加工而成熟在它们参与蛋白质合成之前发生的事件包括末端修剪。生成正确的 5' 和 3' 末端，进行化学修饰以确保正确的结构和功能，并添加最终连接氨基酸的 3' CCA 尾部。此外，不同数量的 tRNA 还含有内含子，这些内含子会中断反密码子；序列并因此使 tRNA 失去功能，因此剪接对于 tRNA 至关重要。 tRNA 内含子的剪接与 mRNA 剪接的不同之处在于它涉及多个因素。蛋白质催化步骤，包括剪接特异性核酸内切酶的内含子切割，然后是外显子末端成熟并通过三功能连接酶（三个的融合体）连接不同的酶域，取决于生物体），最后去除在锥虫中，有一种由先前的活动留下的“悬挂”磷酸盐。含有单个内含子的 tRNA，tRNATyr，负责解码所有酪氨酸密码子。迄今为止，人们对早期潜水中 tRNA 剪接的机制知之甚少我们的研究已经确定了 tRNA 每个步骤的假定成分。剪接反应，每个都具有不寻常的特征，内含子在多个位置被编辑。剪接又需要编辑。剪接核酸内切酶有一个亚基。其他系统中缺少一个关键子单元的结构。连接酶形成更高级的复合物，表明它与其他因素相关。磷酸转移酶具有不寻常的核酸外切酶结构域，表明存在额外的该提案将重点关注每个内含子的表征。 T. brucei 中 tRNA 剪接途径的组成部分，还将探索鉴于 T. brucei 机制是内含子编辑对剪接特异性的贡献。充满独特的功能，成功完成这些研究将产生关于 tRNA 剪接机制和进化的重要基本信息，目前尚不清楚了解原生生物领域。！