DIVERSITY IN THE NUMBERS,TYPES AND SEQUENCES OF TRNA'S

蛋白质的合成依赖于一个复杂的细胞机器，包括转移RNA(TRNA)、氨基酰-tRNA合成酶、起始、延伸和释放因子，以及核糖体RNA(RRNA)和核糖体蛋白。因为翻译是一个基本的细胞过程，强烈的稳定化选择预计会影响其组成部分。然而，基因组测序项目的结果表明，尽管翻译的一般过程在整个进化过程中是保守的，但生物以许多不同的方式保持准确和高效的蛋白质合成。特别是，生物内部和生物之间在tRNAs的数量和类型以及序列和结构特征方面存在着意想不到的差异。因此，过渡机构的弹性显然比人们通常认为的要大。拟议的实验是基于这些观察结果，旨在回答三个相关问题。同种异构体之间序列差异的机制是什么？是什么机制使生物体在进化过程中丢失了“必要的”tRNA基因？细胞所能耐受的tRNA功能受损程度是多少？这项拟议的研究使用生化和遗传学方法来回答这些问题。它将研究(I)反密码子在tRNA功能和进化中的作用；(Ii)补偿tRNA基因丢失的机制；以及(Iii)细胞存活和生长所必需的tRNA功能水平。这些结果将阐明tRNAs中决定其在蛋白质合成过程中接受和捐赠氨基酸功能的元素。此外，他们还将阐明影响tRNAs这些活动的翻译装置的性质。因此，这些结果将与一个普遍的问题相关，即由于细胞内的环境，不同的表型在多大程度上来自单一的基因。对这种现象的理解对于解释DNA序列信息和开发一些已提出的通过操纵翻译机制来控制细菌感染的策略是至关重要的。

Protein synthesis depends on a complex cellular machine comprised of transfer RNA (tRNA), aminoacyl-tRNA synthetases, initiation, elongation, and release factors, as well as ribosomal RNA (rRNA) and ribosomal proteins. Because translation is an essential cellular process, strong stabilizing selection is expected to influence its components. However, results from the genome sequencing projects show that although the general process of translation is conserved throughout evolution, organisms maintain accurate and efficient protein synthesis in many different ways. In particular, there are unexpected differences within and among organisms in the numbers and types as well as in the sequence and structural features of their tRNAs. Thus there is apparently more resiliency in the transitional apparatus than is typically thought. The proposed experiments are motivated by these observations and are designed to answer three related questions. What are the mechanisms that account for sequence variation among isoacceptors? What are the mechanisms that allow organisms to lose "essential" tRNA genes during evolution? What is the extent of impairment in tRNA function that can be tolerated by the cell? The proposed research uses biochemical and genetic approaches to answer these questions. It will examine (i) the role of the anticodon in tRNA function and evolution; (ii) the mechanisms that compensate for tRNA gene loss; and (iii) the levels of tRNA function that are necessary for cell viability and growth. The results will elucidate elements in tRNAs that dictate their amino acid accepting and donating functions during protein synthesis. In addition, they will elucidate the properties of the translational apparatus that influence these activities of tRNAs. Thus the results will be pertinent to the general problem of the extent to which different phenotypes are expressed from a single genotype due to circumstances in the cell. An understanding of this phenomenon is critical to the interpretation of DNA sequence information and to the development of some strategies that have been proposed for controlling bacterial infections by manipulating the translation machinery.