BIOCHEMISTRY OF NOVEL SMALL RNAS IN E COLI AND YEAST

研究的总体目标是制定一个详细的 了解E. 大肠杆菌和酵母菌。 特别感兴趣的是代谢稳定的 E.大肠杆菌和小核RNA（snRNA） 酿酒酵母 每个RNA的主要焦点是 发现并定义其生物学功能。 早期证据 指出4.5S RNA在翻译中的作用， snRNA种类（snR 128）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， 这一点也将受到关注。 一个通用的计划已经开发出来，用于识别小RNA 这是基于遗传和生化策略的功能。 方法包括：1）评价 个体小RNA损失，2）通过RNA的功能作图， 突变，3）未连接RNA负突变的表征 抑制突变体，以识别相互作用的分子，4） 小RNA结合蛋白抗体的制备及应用 这些探针干扰功能和表征天然的 RNA-蛋白质复合物。 提出了E. coli 4.5S RNA的主要作用是：1）鉴定 特异性4.5S RNA结合蛋白，2）表征4.5S 翻译中的RNA要求，3）开发一个功能图谱， 4.5S RNA和4）表征的溶液结构， 4.5S RNA分子。 结构研究将包括扫描 微量热法和质子NMR。 对于snRNA，目的是：1）鉴定RNA的作用。 必需snR 128（由128个核苷酸组成），2）以鉴定 与剪接体的snRNA结合的蛋白质， 描述剪接体snRNP在mRNA成熟中的作用 3）描述新的snRNA种类及其基因。 早期 snR 128物种的重点将是a）评估 与两个内含子结构域互补序列重要性 B）表征三个基因外SNR 128 抑制基因似乎与已知的 mRNA剪接所需的（即RNA基因座）和c） 表征snR 128结合蛋白。 拟议研究的结果应该揭示重要的见解 小RNA在其他细胞中的生物学作用， 描述这些重要物种的一般方法 生物，包括人类。

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.