In vitro Mutagenesis of E. coli Ribosomal Proteins

体外诱变已被广泛用于探测大肠杆菌核糖体RNA的结构和功能，直到最近才将这种强大的技术应用于核糖体蛋白的研究。本计划拨款的目的是修改一个基于T7 RNA聚合酶的双质粒诱导表达系统，该系统适用于大肠杆菌核糖体蛋白L2的研究。该系统通过在两种不同温度下复制电镀变形子，使快速识别显性负突变成为可能。在这项研究中进行的改进是突变核糖体蛋白基因和编码抗生素耐药性的rRNA操纵子的偶联表达。在T7 RNA聚合酶表达系统的诱导下，绝大多数新生核糖体在亚基组装过程中将包括抗生素耐药性标记物以及靶向突变核糖体蛋白。在诱导前组装的野生型核糖体将在体外活性测定期间通过包含适当的抗生素来消除功能。这将允许在体内组装的突变核糖体亚基在体外对其在蛋白质合成的部分反应中的活性/不活性进行表征，从而消除了物理分离两个核糖体种群的需要。计划拨款用于测试一种新技术对核糖体rna -蛋白质相互作用研究的适用性。这项工作的完成将有助于我们更好地了解蛋白质在所有细胞中合成的机制。

In vitro mutagenesis has been used extensively in probing the structure and function of ribosomal RNA from the bacterium E. coli Only recently has this powerful technique been applied toward the study of ribosomal proteins. The objective of this planning grant is the modification of a two plasmid, inducible expression system based upon T7 RNA polymerase that was adapted for an investigation of E. coli ribosomal protein L2. This system has made it possible to quickly identify dominant-negative mutations by replica plating transformants at two different temperatures. The refinements being carried out in this study is the coupled expression of a mutant ribosomal protein gene and an rRNA operon that codes for an antibiotic resistance. Upon induction of the T7 RNA polymerase expression system, the vast majority of de novo ribosomes will include of an antibiotic resistance marker as well as the targeted mutant ribosomal protein during subunit assembly. Wild type ribosomes assembled prior to induction will be functionally eliminated by the inclusion of the appropriate antibiotic during invitro activity assays. This will allow for mutant ribosomal subunits assembled in vivo to be characterized in vitro for their activity/inactivity in the partial reactions of protein synthesis, eliminating the need to physically separate the two ribosome populations. %%% The planning grant is testing the applicability of a new technique to the study of ribosomal RNA-protein interactions. Completion of the work will help us better understand the mechanism by which proteins are synthesized in all cells.