In vitro Mutagenesis of E. coli Ribosomal Proteins

体外诱变技术已被广泛应用于探索细菌核糖体RNA的结构和功能，直到最近才将这一强大的技术应用于核糖体蛋白的研究。这项计划拨款的目标是修改一个基于T7RNA聚合酶的双质粒可诱导表达系统，该系统适用于对大肠杆菌核糖体蛋白L2的研究。这一系统使得在两种不同温度下通过复制电镀转化子快速鉴定显性-负性突变成为可能。在这项研究中进行的改进是突变的核糖体蛋白基因和编码抗生素耐药性的rRNA操纵子的耦合表达。在T7RNA聚合酶表达系统的诱导下，绝大多数从头核糖体将包括一个抗生素耐药标记以及亚基组装过程中的靶向突变核糖体蛋白。在体外活性测定中加入适当的抗生素，在诱导前组装的野生型核糖体将在功能上被消除。这将使在体内组装的突变核糖体亚基能够在体外表征它们在蛋白质合成的部分反应中的活性/不活性，从而消除了物理上分离两个核糖体群体的需要。计划拨款正在测试一项新技术在研究核糖体RNA-蛋白质相互作用方面的适用性。这项工作的完成将有助于我们更好地理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.