CAREER: Mechanistic Investigation of Nucleoside Hypermodification in tRNA

小行星9733746 技术 转运RNA（transfer RNA，tRNA）的修饰是遗传密码表达的基础，其重要性体现在系统发育的保守性和丰富的结构多样性上。本研究的目的是了解生物合成的肌苷，一个修饰的核苷普遍存在于整个细菌和真核生物。鸟苷具有不寻常的7-脱氮鸟苷核心结构，特异性地出现在编码天冬酰胺、天冬氨酸、组氨酸和酪氨酸的tRNA的摆动位置，这表明其在调节翻译保真度和/或效率中的作用。更具体地说，涉及酶S-腺苷甲硫氨酸：tRNA核糖基转移酶异构酶（QueA）的分子机制，它催化的倒数第二个步骤的肌苷生物合成和核糖基的转移，从辅因子S-腺苷甲硫氨酸（ADMET）的肌苷-tRNA前体进行了研究。实验包括纯化的重组酶的区域和立体化学以及与天然底物和类似物的反应动力学的研究，并进行结构研究。还研究了7-脱氮鸟苷类超修饰tRNA核苷的生物合成和功能，如鸟苷的糖基化衍生物和超修饰核苷古苷。 教育的重点是鼓励本科生通过荣誉研究论文计划参与生物化学研究，这是一个试点计划，试图取代和振兴目前的部门荣誉学位要求。 2.非技术性。 涉及转移RNA（tRNA）的分子相互作用是遗传密码表达的基础。 正是在氨基酸与tRNA的正确连接以及随后通过信使RNA（mRNA）与tRNA的密码子-反密码子配对的蛋白质合成中，遗传信息作为蛋白质的物理表现得以实现。tRNA的一个典型结构特征是修饰核苷的惊人多样性，其中许多在不同的生物体中高度保守。 修饰的范围可以从相对较小的变化到复杂的过度修饰，这导致核苷的根本结构变化，需要多个步骤才能完成。这项研究构成了一套广泛的实验方法，旨在详细了解超修饰核苷肌苷生物合成中的关键步骤，该步骤仅发生在编码天冬酰胺、天冬氨酸、组氨酸和酪氨酸氨基酸的tRNA的反密码子的第一个位置（摆动位置）。其在反密码子中的特定位置表明其在调节蛋白质合成的保真度和/或效率中的作用，这需要进一步的实验支持。教育部分的重点是通过设计和实施荣誉研究论文计划，以测试建立一个一般要求的可行性，而不是目前的部门荣誉学位，为该部门提供的化学和生物化学学位，增加本科生在生物化学研究中的参与。

9733746 Iwata-Reuyl TECHNICAL Modification of transfer RNA (tRNA) is fundamental to expression of the genetic code, its apparent importance is implied by both the phylogenetic conservation and the rich structural diversity. This study is designed to understand the biosynthesis of queuosine, a modified nucleoside ubiquitous throughout the Bacteria and Eukarya. Queuosine possesses an unusual 7-deazaguanosine core structure, occurs specifically at the wobble position of tRNAıs coding for asparagine, aspartic acid, histidine, and tyrosine, which suggests a role in modulating translational fidelity and/or efficiency. More specifically, the molecular mechanism involving the enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step of queuosine biosynthesis and the transfer of ribosyl from the co-factor S-adenosylmethionine (AdoMet) to a queuosine-tRNA precursor is studied. The experiments include studies of purified recombinant enzymes for their regio- and stereochemistry and reaction kinetics with the natural substrates and analogs, and do structural studies. The biosynthesis and function of the 7-deazaguanosine class of hypermodified tRNA nucleosides, such as glycosylated derivatives of queuosine and the hypermodified nucleoside archaeosine is also studied. The educational focus is placed on encouraging the participation of undergraduates in biochemical research through an honors research thesis program, which is a pilot program with an attempt to replace and to revitalize the current departmental honors degree requirements. 2. NON-TECHNICAL. Molecular interactions involving transfer RNA (tRNA) are fundamental to expression of the genetic code. It is in the correct attachment of amino acids to tRNA, and subsequent protein synthesis through codon-anticodon pairing of the messenger RNA (mRNA) with the tRNA, that the physical manifestation of genetic information as protein is realized. A characteristic structural feature of tRNA is the presen ce of an astonishing diversity of modified nucleosides, many of which are highly conserved among disparate organisms. Modification can range from relatively minor changes to complex hypermodification, which results in radical structural changes to the nucleoside and requires multiple steps to complete. This study constitutes a broad set of experimental approaches designed to develop a detailed understanding of a critical step in the biosynthesis of the hypermodified nucleoside queuosine, which occurs exclusively at the first position (wobble position) of the anticodon in tRNAıs coding for the amino acids asparagine, aspartic acid, histidine, and tyrosine. Its specific location in the anticodon suggests a role in modulating the fidelity and/or efficiency of protein synthesis, for which further experimental support is needed. The educational component focuses on increasing the participation of undergraduates in biochemical research through the design and implementation of an honors research thesis program, which serves to test the feasibility of instituting a general requirement, in lieu of the current departmental honors degree, for the Chemistry and Biochemistry degrees offered by the department.