BIOCHEMISTRY OF NOVEL SMALL RNAS IN E COLI AND YEAST

所提出的研究的总体目标是制定详细的 对新的小RNA物种在E. 大肠杆菌和酵母菌。特别令人感兴趣的是代谢稳定 大肠杆菌4.5S RNA及其小核糖核酸(SnRNA) 酿酒酵母。每个RNA的主要关注点是 发现并定义其生物学功能。早期证据 指出4.5S RNA物种和其中之一在翻译中的作用 SnRNA物种(SnR128)显示出在mRNA中发挥作用的前景 拼接。4.5S RNA具有特别独特的结构和 这也将受到关注。 已经开发出一种识别小RNA的通用计划 基于遗传和生化策略的功能。 这些方法包括：1)评价人体的生理效应 单个小RNA丢失，2)通过以下方式对RNA进行功能定位 诱变；3)去连接RNA负链的鉴定 抑制突变体，以识别相互作用的分子，4) 抗小RNA结合蛋白抗体的制备及应用 干扰这些探针的功能，并将其描述为 RNA-蛋白质复合体。 E.Coli4.5S RNA的具体目的是：1)鉴定 特异的4.5S RNA结合蛋白，2)4.5S的特性 翻译中的RNA需求，3)开发一个功能图谱 4.5S RNA和4)表征其溶液结构 4.5S RNA分子。结构研究将包括扫描 微量热法和质子核磁共振。 对于SnRNA来说，其目的是：1)确定 必需的SnR128(由128个核苷酸组成)，2)识别 与剪接体的SnRNA结合的蛋白质和 剪接体SnRNPs在mRNA成熟中的作用 3)描述新的SnRNA物种及其基因。早些时候 对SNR128物种的重点将放在a)评估 两个内含子结构域互补序列的重要性 剪接所需，b)表征三个外源SNR128 抑制子似乎对应于已知的 信使核糖核酸剪接所需(即RNA基因座)和c) 鉴定SnR128结合蛋白。 拟议的研究结果应该会揭示出重要的见解 小RNA在其他细胞中的生物学作用，并建立 在《高等医学》中描述这些重要物种的一般方法 包括人类在内的有机体。

The overall goal of the research proposed is to develop a detailed understanding about the biology of novel small RNA species in E. coli and yeast. Of special interest are the metabolically stable 4.5S RNA of E. coli and the small nuclear RNAs (snRNA) of Saccharomyces cerevisiae. the major focus for each RNA is discovering and defining its biological function. Early evidence points to a role in translation for the 4.5S RNA species and one of the snRNA species (snR128) shows promise for a role in mRNA splicing. The 4.5S RNA has a particularly unique structure and this will also receive attention. A universal plan has been developed for identifying small RNA function that is based on both genetic and biochemical strategies. The approaches include: 1) evaluating the physiological effects of individual small RNA loss, 2) functional mapping of the RNAs by mutagenesis, 3) characterization of unlinked RNA-minus suppressor mutants, to identify interacting molecules, 4) preparation of antibodies to small RNA binding proteins and use of these probes to interfere with function and characterize native RNA-protein complexes. The specific aims for the E. coli 4.5S RNA are: 1) to identify specific 4.5S RNA binding proteins, 2) to characterize the 4.5S RNA requirement in translation, 3) to develop a functional map of the 4.5S RNA and 4) to characterize the solution structure of the 4.5S RNA molecule. The structural studies will include scanning microcalorimetry and proton NMR. For the snRNAs the aims are: 1) to identify the role of the essential snR128 (consisting of 128 nucleotides), 2) to identify proteins that bind to the snRNAs of the spliceosome and characterize the role of spliceosome snRNPs in mRNA maturation and 3) to describe new snRNA species and their genes. Early emphasis with the snR128 species will be on a) assessing the importance of sequences complementary to two intron domains required for splicing, b) characterizing three extragenic SNR128 suppressors that appear to correspond to genes known to be required for mRNA splicing (i.e. the rna loci) and c) characterizing snR128 binding proteins. Results from the proposed studies should reveal important insights into the biological roles of small RNAs in other cells and establish general approaches for characterizing these vital species in higher organisms, including humans.