Modified Uridines, Contributors of Novel Chemistries to Functional RNA

基因组信息的解码是所有细胞中蛋白质合成的生物学基础，看似如此简单，但现在却变得复杂起来，因为它需要对涉及的RNA分子进行一系列不同的化学添加，即转移RNA(TRNAs)。TRNAs负责将蛋白质的遗传信息翻译成该蛋白质的氨基酸序列。许多缺乏这些化学修饰的tRNA，特别是在两个不同的位置(反密码子摆动位置-34和/或嘌呤核苷-37)，将不能识别信使RNA中包含的氨基酸编码。TRNAs中这两个位置的化学修饰挽救了tRNAs读取氨基酸密码的能力。有两个密码子的氨基酸的tRNA在阅读时必须受到限制，这样它们就不会阅读其他氨基酸的密码子。有四个密码子的氨基酸的tRNA必须具有阅读这四个密码子的能力。值得注意的是，这两组tRNA的修饰方式不同。因此，该提案的长期目标是：a)建立依赖于tRNA修饰的密码子解码的一般原则；b)通过将教育与科学相结合，教育和激发年轻科学家对RNA化学的了解；c)教育科学界，使他们了解修饰tRNA功能的重要性；以及d)将对修饰结构/功能关系的理解应用于设计分子和细胞生物学中的RNA工具。这项研究集中在物理化学机制(S)，通过修改可能控制tRNA的识别两个密码子而不是四个密码子。明显不同的修饰是否通过限制前者的密码子识别和扩大后者的摆动来帮助tRNA解码两个密码子而不是四个密码子的能力？密码子识别需要蛋白质合成机制，即核糖体，诱导正确的适合tRNA的结构吗？修饰是否为蛋白质合成预先形成了tRNA结构？为了回答第一个问题，将限制tRNA与两个密码子结合的修饰与不同修饰的tRNA与核糖体上四个密码子的结合进行比较。为了回答第二和第三个问题，各种修饰对tRNA稳定性、溶液结构和核糖体结构的影响将与它们的解码能力有关。核磁共振波谱的溶液结构将相互比较，并与核糖体上密码子结合的结构进行比较。这些实验将确定修饰是否在密码子结合之前预先构建tRNA的结构，以及核糖体是否诱导密码子结合的结构。理解tRNA阅读遗传密码的修饰依赖机制是生物化学、细胞生物学和年轻科学家教育的基础。一种新的基于结构的核糖体摆动解码模型将被产生，该模型具有依赖于修饰的、新的碱基配对几何结构。该模型将包括依赖修饰的预形成或核糖体诱导适合的反密码子体系结构。该项目的更广泛影响集中在教育目标上，即向年轻人的头脑中灌输科学好奇心，并通过激发年轻人参与回答有关RNA生物学的问题来获得研究经验。通过将研究与教育相结合，北卡罗来纳州立大学和北卡罗来纳农业技术州立大学的本科生和研究生将从同行和导师那里学习科学方法。为了造福社会，代表不足的少数群体和妇女将接受物理生物化学方面的指导；他们在该系目前的专业中占很大比例(12%)。NCSU的研究基础设施将通过购买用于RNA物理化学研究的微量热计而得到加强。一些仪器的集体住房将建立一个共享的教育基础设施，并为致力于RNA研究的三角大学之间的互动和指导创造一个支持性的环境。

Decoding of genomic information, so basic to the biology of protein synthesis in all cells and seemingly so simple, now is complicated by the finding that it requires a diverse set of chemical additions to the RNA molecules that are involved, transfer RNAs (tRNAs). tRNAs are responsible for translating the genetic information for a protein into the amino acid sequence of that protein. Many tRNAs devoid of these chemical modifications, particularly at two different positions (the anticodon wobble position-34 and/or the purine nucleoside-37), will not recognize the amino acid code incorporated into messenger RNA. The chemical modifications at these two positions in the tRNAs rescue tRNAs' ability to read the amino acid codes, codons. The tRNAs for amino acids that have two codons have to be restricted in their reading so that they do not read the codons of other amino acids. The tRNAs for amino acids that have four codons have to have the ability to read the four codons. Significantly, these two sets of tRNAs are differently modified. Thus, the proposal's long-term objectives are to a) Establish general principles for tRNA's modification-dependent decoding of codons; b) Educate and excite young scientists to the chemistries of RNA by integrating education with science; c) educate the scientific community as to the importance of modifications tRNA function; and d) apply an understanding of modification structure/function relationships to designing RNA tools in molecular and cellular biology. The research is focused on the physical chemical mechanism(s) by which modifications may control tRNA's recognition of two versus four codons. Do distinctly different modifications contribute to tRNA's ability to decode two versus four-fold codons by restricting codon recognition in the former and expanding wobble in the latter? Does codon recognition require that the protein synthesizing machinery, the ribosome, induce a correct fit architecture to tRNA? Do modifications pre-form the tRNA structure for protein synthesis? To answer the first question modifications that restrict tRNAs binding to two codons will be compared to the binding of differently modified tRNAs to four codons on the ribosome. To answer the second and third questions, the affects of the various modifications tRNA stability, and structure in solution, and structure on the ribosome will be related to their ability to decode. Solutions structures from nuclear magnetic resonance spectroscopy will be compared to each other and to the structures bound to codon on the ribosome. These experiments will determine whether modifications pre-structure the tRNA prior to codon binding, and whether the ribosome induces architecture for codon binding. An understanding of the modification-dependent mechanism of tRNA's reading of the Genetic Code is fundamental to biochemistry, cell biology and to the education of young scientists. A new structural-based model of wobble decoding on the ribosome with modification-dependent, novel base pairing geometries will be generated. The model will include either a modification-dependent pre-formed or ribosome induced-fit anticodon architecture.The broader impact of the project is focused on the educational objective to instill scientific curiosity in young minds, and research experience in young hands by exciting their involvement in answering questions about RNA biology. By integrating research with education undergraduate and graduate students at North Carolina State University and North Carolina Agricultural and Technical State University, a historically black college, will learn scientific methodology from peers and mentors. To the benefit of society underrepresented minorities and women will be mentored in physical biochemistry; they represent a significant proportion (12%) of the Department's present majors. NCSU's research infrastructure will be enhanced by purchase of a microcalorimeter for the physiochemical study of RNA. The collective housing of a number of instruments will establish a shared educational infrastructure and creates a supportive environment for interactions and mentoring among Triangle Universities dedicated to RNA research.