STRUCTURE/FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

描述（改编自申请人摘要）：本研究的目的 建议是了解：（1）结构和 E.大肠杆菌启动子tRNA和（2） 分子机制的基础上的具体相互作用的组成部分， 翻译机器 一个重要的问题是高度特异性的 通过Met-tRNA转化酶识别tRNA。 的组合 将采用结构、生物化学和遗传学方法对此进行研究。 这些包括（I）NMR光谱分析tRNA的结构 底物，（ii）晶体学，以确定蛋白质的结构 和tRNA-蛋白质复合物，（iii）拓扑结构的研究 使用交联实验的蛋白质与tRNA的相互作用， 保护实验和通过检查蛋白质中的抑制突变， 弥补突变tRNA甲酰基化缺陷，和（iv） 位点特异性诱变以鉴定蛋白质中重要的氨基酸 tRNA的选择和功能。 类似的方法将用于研究tRNA与其他蛋白质的相互作用。 蛋白质，特别是抑制因子的体内选择和分析 IF2、IF3或其他染色体基因突变。 这样的工作可以提供 引发因子和引发剂之间相互作用的信息 tRNA，并可能导致识别参与翻译的新基因， 入会仪式 翻译中间体的识别工作 将继续体内启动。 具体关注的问题是：（一） IF2是核糖体fMet-tRNA的载体吗？ (ii)30S核糖体 首先与起始tRNA结合，然后与mRNA结合，或者反之亦然？ （三） 翻译再起始的起始tRNA的要求是否 与从头开始相同？ 最后，All：U24碱基对的作用 真细菌启动子tRNA在启动中的独特作用将沿着 为什么将碱基对引入E.大肠延长器 甲硫氨酸tRNA可防止tRNA在体内积累。

DESCRIPTION (Adapted from applicant's abstract): The objectives of this proposal are to understand: (1) the relationship between the structure and the many distinctive properties of E. coli initiator tRNA and (2) the molecular mechanisms underlying its specific interactions with components of the translational machinery. An important question is the molecular basis of the highly specific recognition of the tRNA by Met-tRNA transformylase. A combination of structural, biochemical, and genetic approaches will be used to study this. These include (I) NMR spectroscopy to analyze the structure of the tRNA substrate, (ii) crystallography to determine the structure of the protein and the tRNA-protein complex, (iii) investigation of the topology of interaction of the protein with the tRNA using crosslinking experiments, protection experiments and by examining suppressor mutations in the protein that compensate for defects in formylation of mutant tRNAs, and (iv) site-specific mutagenesis to identify amino acids important in the protein for tRNA selection and function. Similar approaches will be used to study interactions of tRNA with other proteins, in particular, in vivo selection and analysis of suppressor mutations in IF2, IF3, or other chromosomal genes. Such work could provide information on interactions between the initiation factors and the initiator tRNA and could lead to identification of new genes involved in translational initiation. Work on identification of intermediates in translation initiation in vivo will continue. Questions of specific interest are: (I) is IF2 a carrier of fMet-tRNA to the ribosome? (ii) Does the 30S ribosome bind first to the initiator tRNA and then to the mRNA or vice versa? (iii) Are the requirements in an initiator tRNA for translational reinitiation the same as for de novo initiation? Finally, the role of the All:U24 base pair unique to eubacterial initiator tRNAs in initiation will be studied along with questions of why introduction of the base pair to an E. coli elongator methionine tRNA prevents accumulation of the tRNA in vivo.