RIBOSOMAL PEPTIDYL TRANSFERASE CENTER

多肽键的形成是蛋白质合成的中心反应之一。它是由位于大核糖体亚基上的肽基转移酶中心催化的。尽管多年来的研究，但对肽基转移酶的组成、结构和功能仍不清楚。理解多肽转移酶中心的组织和功能的主要障碍之一是核糖体的巨大复杂性。因此，我们提出了一系列实验，其目标是通过显著降低肽基转移酶活性亚核糖体颗粒的复杂性来分离基本肽转移酶。对嗜热真细菌Thermus Aquaticus的50S核糖体亚基进行广泛的蛋白质提取，形成了含有23S rRNA、5S rRNA和仅8个核糖体蛋白的颗粒(KSP颗粒)，具有较高的肽基转移酶活性。通过用从KSP颗粒中分离出的不同蛋白质组合重组rRNA，可以减少KSP颗粒的蛋白质组成。将确定支持肽基转移酶活性所需的最小蛋白质集。接下来，我们将研究5S rRNA对重建活性粒子的重要性。初步研究表明，与23S rRNA的两个结构域相互作用的小分子药物可以取代与23S rRNA中较远的结构域相互作用的5S，5S的存在对功能肽转移酶的体外组装是必不可少的。将研究在抗生素或小的5S rRNA片段存在下重建的5S缺陷颗粒恢复高水平肽转移酶活性的可能性。接下来，23S rRNA的延伸片段将被删除，这对肽转移酶的催化不是必需的。这些实验将基于最近的一项发现，即功能活跃的大核糖体亚基可以与体外转录的23S rRNA重组。核糖体蛋白在肽基转移酶活性中的作用可能与稳定催化活性rRNA的功能结构有关。我们将结合使用rRNA突变、体外组装和从亚核糖体颗粒中选择扩增RNA，以筛选rRNA突变，通过稳定rRNA结构，可能弥补核糖体蛋白的缺乏。类似的方法将被用来寻找突变，以弥补催化活性颗粒中缺乏5S rRNA和/或23S rRNA延伸片段的缺失。一旦最低限度的肽基转移酶被分离出来，它的结构、详细的结构和功能将被分析。这些信息不仅可能阐明多肽键形成的催化机制，而且为了解原体的组织和功能以及翻译装置的进化提供了重要的见解。了解核糖体主要催化中心的结构和功能对于合理设计新抗生素和克服耐药机制是必不可少的。

Peptide bond formation is one of the central reactions of protein synthesis. It is catalyzed by the peptidyl transferase center located on the large ribosomal subunit. In spite of years of research, the composition, structure and function of peptidyl transferase remains obscure. One of the main obstacles for understanding organization and function of the peptidyl transferase center is the enormous complexity of the ribosome. Therefore, we propose a set of experiments whose goal is to isolate the elementary peptidyl transferase by significantly reducing complexity of the peptidyl transferase-active subribosomal particles. Subjection of 50S ribosomal subunits from thermophilic eubacterium Thermus aquaticus to extensive protein extraction procedures results in formation of particles (KSP particles) which, while containing 23S rRNA, 5S rRNA and only 8 ribosomal proteins, posses a high level of peptidyl transferase activity. The protein composition of KSP particles will be reduced by reconstituting rRNA with different combinations of proteins isolated from KSP particles. The minimal set of proteins required to support peptidyl transferase activity will be identified. Next, the importance of 5S rRNA for reconstituting active particles will be investigated. Preliminary studies indicated that 5S, which interacts with distant domains in 23S rRNA and whose presence is essential for in vitro assembly of functional peptidyl transferase, can be replaced by small drug molecules also interacting with the same two domains of 23S rRNA. The possibility of restoring high levels of peptidyl transferase activity of 5S-deficient particles reconstituted in the presence of antibiotics or small 5S rRNA fragments will be investigated. Next, extended stretches of 23S rRNA, non-essential for peptidyl transferase catalysis, will be deleted. These experiments will be based on a recent finding that functionally active large ribosomal subunits of T. aquaticus can be reconstituted with the in vitro transcribed 23S rRNA. The role of ribosomal proteins in peptidyl transferase activity maybe that of stabilizing the functional structure of catalytically-active rRNA. We will use a combination of rRNA mutagenesis, in vitro assembly and selection-amplification of RNA from subribosomal particles in order to select for rRNA mutations that, by stabilizing rRNA structure, may compensate for the lack of ribosomal proteins. An analogous approach will be used to find mutations compensating for the lack of 5S rRNA and/or the lack of extended segments of 23S rRNA in catalytically active particles. Once the minimalist peptidyl transferase is isolated, its architecture, detailed structure and functions will be analyzed. This information may not only clarify the mechanism of catalysis of peptide bond formation, but also provide important insights into organization and function of the protoribosome and evolution of translation apparatus. Understanding structure and function of the main catalytic center of the ribosome is indispensable for the rational design of new antibiotics and overcoming mechanisms of drug resistance.