BIOSYNTHESIS OF TRANSFER, 4.5S & 6S RIBONUCLEIC ACIDS

一个多部分的研究计划已被设计，以产生信息， 对E.大肠杆菌和生物学作用， 代谢稳定的E. coli 4.5S和6S RNA。 显示代谢调节差异效应的tRNA基因将被 分离并表征了相关的转录控制元件 通过序列和功能分析。 转录特性将 在常见的质粒表达载体中进行比较，以了解 对生长相关控制的不同反应的基础。 独特 涉及控制的结构元件将通过体外修饰， 诱变以产生功能改变的变体用于比较 评价 该计划的一个主要部分将与生物化学有关， 代谢稳定的E. coli 4.5S和6S RNA。 主要重点将 发现和表征这些生物学功能 小RNA 我们已经证明，4.5S RNA是生长所必需的， 蛋白质的合成在无法产生 了 6S RNA在类似于“信号”的复合物中的出现 动物细胞的“识别颗粒”表明了这一可能的作用 蛋白质转运的物种。 功能研究的策略 这些RNA将：1）识别和表征主要的 当RNA的合成被选择性阻断时受影响的功能，2） 开发和表征4.5S或6S RNA功能改变的突变体 和3）鉴定4.5S和6S RNA结合蛋白，并表征其 合成和功能。 最后，将启动4.5S和6S RNA的结构研究， 为最终的结构-功能研究做准备， 需要解决新的RNA结构。 重组克隆的存在 其过量产生这些RNA达30-50倍， 特征可能。 4.5S RNA的初步结果表明， 其二级结构可以是近乎完美发夹螺旋。 计划 结构分析，许多将与其他机构合作进行， 将包括：a）S1核酸酶图谱，B）高分辨率质子磁共振成像， 共振光谱，c）激光散射，d）微量热法，e） 圆二色性和f）X射线晶体学。

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.