Ribosomal RNA Catalyzed Protein Synthesis

核糖体是所有生物体内形成多肽键的催化剂。核糖体的50S核糖体亚基含有多肽基转移酶中心，在那里形成了多肽键。最近完整的50S亚基的高分辨晶体结构表明，肽基转移酶中心完全由rRNA组成。活性部位内的单核苷酸具有特殊的化学碱性，这可能使其成为形成多肽键的普通酸碱催化剂。这意味着核糖体是一种RNA酶或核酶，尽管这一点尚未得到生化证明。这个项目的具体目标是在没有核糖体蛋白的情况下，通过体外转录的23S rRNA来证明肽基转移酶的活性。为了实现这一目的，将使用一种新版本的肽基转移酶反应，其缓解了先前使用的片段反应所需的人工反应条件。50S亚基的结构将被用来设计截短的23S rRNA片段，这些片段可能折叠成活性构象。这些实验能够提供确凿的生化证据来确定23S rRNA是否是蛋白质合成的催化剂，并将为分析核糖体反应机制提供一个生化顺从的系统。

0096772StrobelThe ribosome is the catalyst of peptide bond formation within all living organisms. The 50S ribosomal subunit of the ribosome contains the peptidyl transferase center where the peptide bonds are formed. The recent high resolution crystal structure of the intact 50S subunit suggests that the peptidyl transferase center is composed exclusively of rRNA. The unusual chemical basicity of a single nucleotide within the active site may allow it to serve as a general acid-base catalyst for peptide bond formation. This implies that the ribosome is an RNA enzyme or ribozyme, though this has yet to be biochemically demonstrated. The specific objective of this project is to demonstrate peptidyl transferase activity by an in vitro transcribed 23S rRNA in the absence of ribosomal proteins. To achieve this objective a new version of the peptidyl transferase reaction that alleviates the artifactual reaction conditions required of the previously used fragment reaction will be utilized. The structure of the 50S subunit will be employed to design truncated 23S rRNA fragments that are likely to fold into an active conformation. These experiments are capable of providing definitive biochemical evidence to establish whether 23S rRNA is the catalyst of protein synthesis and will provide a biochemically amenable system for the analysis of the ribosomal reaction mechanism.