BIOSYNTHESIS OF TRANSFER, 4.5S & 6S RIBONUCLEIC ACIDS

已经设计了一个由多个部分组成的研究计划，以获得关于 大肠杆菌中tRNA生物合成的调控及其生物学作用 代谢稳定的大肠杆菌4.5S和6S RNA的结构 表现出不同代谢调节效应的tRNA基因将是 分离并鉴定相关的转录调控元件 通过序列分析和功能分析。转录属性将 在通用的质粒表达载体中进行比较，以深入了解 对与生长相关的控制的不同反应的基础。独一无二 对照中涉及的结构元件将在体外被修饰 突变产生功能改变的变异体用于比较 评估。 该计划的主要部分将与生物化学有关 代谢稳定的E.Coli4.5S和6S RNA。主要重点将是 致力于发现和表征这些物质的生物学功能 小RNA。我们已经证明了4.5S RNA对生长和 这种蛋白质的合成在不能产生蛋白质的细胞中严重受损 它。6S RNA出现在一个类似于‘信号’的复合体中 动物细胞的识别颗粒表明了这一现象的可能作用 蛋白质易位的物种。研究生态文明功能的策略 这些RNA将：1)识别和表征主要的 选择性阻断核糖核酸合成影响功能(S)，2) 4.5S或6S RNA功能突变株的选育与鉴定 3)鉴定4.5S和6S RNA结合蛋白 综合和功能。 最后，将启动4.5S和6S RNA的结构研究，以 为最终的结构-功能研究做准备，并认识到 需要解决新的RNA结构。重组克隆的存在 将这些RNA的产量提高了30-50倍，这使得详细的生物物理 有可能进行定性。4.5S RNA的初步结果表明 它的二级结构可能是一个近乎完美的发夹螺旋。计划中的 结构分析，其中许多是与其他人合作执行的， 将包括：a)S1核酸酶图谱，b)高分辨率质子磁性 共振光谱，c)激光光散射，d)微量热法，e) 圆二色谱和f)x射线结晶学。

A multi-part research program has been designed to yield information about the regulation of tRNA biosynthesis in E. coli and the biological roles and structures of the metabolically stable E. coli 4.5S and 6S RNAs. tRNA gens showing differential effects of metabolic regulation will be isolated and the associated transcriptional control elements characterized by sequence and functional analyses. The transcriptional properties will be compared in a common plasmid expression vector to gain insight into the bases for the different responses to growth-related control. Unique structural elements implicated in control will be modified by in vitro mutagenesis to produce functionally altered variants for comparative evaluation. A major portion of the program will be concerned with the biochemistry of the metabolically stable E. coli 4.5S and 6S RNAs. Primary emphasis will be on discovering and characterizing the biological functions of these small RNAs. We have shown that the 4.5S RNA is essential for growth and that protein synthesis is seriously compromised in cells unable to produce it. The occurrence of the 6S RNA in a complex resembling the 'signal recognition particle' of animal cells suggests a possible role for this species in protein translocation. The strategy in studying the function of these RNAs will be: 1) to identify and characterize the primary function(s) affected when synthesis of the RNA is blocked selectively, 2) to develop and characterize mutants with altered 4.5S or 6S RNA function and 3) to identify 4.5S and 6S RNA binding proteins and characterize their synthesis and function. Finally, structural studies of the 4.5S and 6S RNAs will be initiated to prepare for eventual structure-function studies and in recognition of the need to solve new RNA structures. The existence of recombinant clones which overproduce these RNAs by 30-50 fold makes detailed biophysical characterization possible. First results with the 4.5S RNA indicate that its secondary structure may be a near-perfect hairpin helix. The planned structural analyses, many to be performed in collaboration with others, will include: a) S1 nuclease mapping, b) high resolution proton magnetic resonance spectroscopy, c) laser light scattering, d) microcalorimetry, e) circular dichroism and f) x-ray crystallography.