Modified Uridines, Contributors of Novel Chemistries to Functional RNA

基因组信息的解码，对于所有细胞中蛋白质合成的生物学来说是如此的基础，看起来如此简单，现在却变得复杂了，因为它需要在涉及的RNA分子上添加一系列不同的化学物质，即转移RNA （tRNAs）。trna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），将无法识别整合到信使RNA中的氨基酸代码。trna中这两个位置的化学修饰挽救了trna读取氨基酸密码子的能力。有两个密码子的氨基酸的trna在读取时必须受到限制这样它们就不能读取其他氨基酸的密码子。具有四个密码子的氨基酸的trna必须具有读取四个密码子的能力。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tRNA修饰依赖性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化学的认识；c)教育科学界认识到修饰tRNA功能的重要性；d)运用对修饰结构/功能关系的理解来设计分子和细胞生物学中的RNA工具。研究的重点是物理化学机制(s)的修饰可能控制tRNA的两个或四个密码子的识别。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促进tRNA解码两倍或四倍密码子的能力？密码子识别是否需要蛋白质合成机制，即核糖体，诱导正确的适合tRNA的结构？修饰是否预先形成蛋白质合成的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。为了回答第二个和第三个问题，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，并与核糖体上与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了tRNA，以及核糖体是否诱导了密码子结合的结构。了解tRNA读取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。一个新的基于结构的模型的摆动解码的核糖体与修饰依赖，新的碱基配对几何将产生。该模型将包括修饰依赖性预形成或核糖体诱导拟合的反密码子结构。该项目更广泛的影响集中在教育目标上，通过激发年轻人参与回答有关RNA生物学的问题，在年轻人的头脑中灌输科学好奇心，并在年轻人的手中积累研究经验。北卡罗莱纳州立大学和北卡罗莱纳州农业技术大学（一所历史悠久的黑人大学）的本科生和研究生将通过将研究与教育结合起来，从同龄人和导师那里学习科学方法论。为了社会的利益，代表性不足的少数民族和妇女将在物理生物化学方面得到指导；他们占了系里现有专业的很大比例（12%）。通过购买用于RNA物理化学研究的微热量计，NCSU的研究基础设施将得到加强。许多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.