STRUCTURE/FUNCTION OF TRANSFER RIBONUCLEIC ACIDS

描述(改编自申请人的摘要)：本报告的目的 建议是要了解：(1)结构与结构的关系 大肠杆菌启动子tRNA的许多独特性质和(2) 其与不同成分的特异性相互作用的分子机制 翻译机器。 一个重要的问题是高度特异性的分子基础 Met-tRNA转换酶对tRNA的识别。一种组合 将使用结构、生物化学和遗传方法来研究这一点。 其中包括：(1)核磁共振波谱分析tRNA的结构 底物，(Ii)确定蛋白质结构的结晶学 和tRNA-蛋白质复合物，(III)拓扑结构的研究 通过交联实验使蛋白质与tRNA相互作用， 保护实验和通过检测蛋白质中的抑制子突变 以弥补突变tRNAs甲酰化的缺陷，以及(Iv) 定点突变以确定蛋白质中重要的氨基酸 用于tRNA的选择和功能。 类似的方法将被用来研究tRNA与其他 蛋白质，特别是体内抑制子的选择和分析 IF2、IF3或其他染色体基因的突变。这样的工作可以提供 关于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。翻译中的中介语识别研究 体内启动将继续进行。特别感兴趣的问题有：(I) IF2是核糖体的fMet-tRNA载体吗？(Ii)30S核糖体 首先与启动子tRNA结合，然后与mRNA结合，或者反之亦然？(Iii) 启动子tRNA中对翻译重新启动的要求是 和从头开始一样吗？最后，ALL：U24碱基对的作用 优生菌启动子tRNAs独有的启动子将沿着 关于为什么将碱基对引入到大肠杆菌延伸子中的问题 蛋氨酸tRNA可防止tRNA在体内积聚。

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.