DEFINING THE ROLE PLAYED BY 23S RRNA IN TRANSLATION

将细胞中的遗传信息翻译成功能性蛋白质是一项重要且高度保守的功能。核糖体是一种两亚基大分子复合物，由细菌中的三个大rna （RNAs）和50多种蛋白质（r-蛋白）组成，是复杂而协调的翻译过程的催化剂和框架。肽基转移酶，可能是核糖体最原始的活动，是核心的重要性。生命起源的理论预测了rRNA在翻译中的核心作用，这一预测得到了RNA催化作用的证明，并与rRNA序列之间观察到的极端系统发育保守性相一致。大量的生化和遗传学研究表明，rnas在翻译过程中起着主要的功能作用，特别是23S rRNA在中心催化事件肽键形成中起着重要作用。核糖体的结构研究，包括低温电子显微镜和x射线晶体学，正在产生丰富的信息。然而，这种方法产生的核糖体的静态视图将无法揭示复杂的翻译过程的机制或动力学。本文介绍了体外遗传学和生物化学方法来鉴定肽基转移酶（RNA或蛋白质）活性位点的分子成分。这些研究的目标是了解这些成分如何共同形成肽键，并了解这种活性如何在每个催化循环之间得到控制，从而产生高保真编码蛋白产物。首先，将使用定点诱变和修饰干扰方法来鉴定23S rRNA中特异性参与tRNA底物结合和核糖体催化的核苷酸。其次，最近开发的迭代体外选择方法将用于优化由体外转录的嗜脂嗜热芽孢杆菌23S rRNA重组的核糖体的活性。最后，将采用交联、动力学研究和化学修饰等多种生化方法来表征tRNA结合在翻译延伸中的杂化状态的结构和功能。从这些研究中获得的信息将最终有助于确定当前核糖体特异性抗生素的特征，这些特征使它们有效，以及这些抗生素被宿主逃避的机制。最终，这将导致新型抗生素的合理设计。

Translation of the genetic information in the cell into functional proteins is an essential and highly conserved function. The ribosome, a two-subunit macromolecular complex, composed in bacteria of three large RNAs (rRNAs) and more than 50 proteins (r-proteins), is the catalyst and framework for the intricate and coordinated process of translation. Peptidyl transferase, likely the most primitive activity of the ribosome, is of central importance. Theoretical considerations of the origin of life predict a central role for the rRNAs in translation, a prediction strengthened by the demonstration of catalysis by RNA and consistent with the extreme phylogenetic conservation observed among rRNA sequences. Extensive biochemical and genetic studies indicate that the rRNAs play a primary functional role in the processes of translation, and in particular 23S rRNA in the central catalytic event, peptide bond formation. Structural studies of the ribosome, including cryoelectron microscopy and X-ray crystallography, are yielding a wealth of information. However, the static views of the ribosome yielded by such approaches will not reveal the mechanism or dynamics of the intricate process of translation. This proposal describes in vitro genetic and biochemical approaches to identify the molecular components at the active site of peptidyl transferase (RNA or protein). The goal of these studies is to understand how these components conspire in the formation of peptide bonds and to understand how this activity is controlled between each catalytic cycle to allow for the generation of high-fidelity encoded protein products. First, site-directed mutagenesis and modification interference approaches will be used to identify nucleotides in 23S rRNA that are specifically involved in tRNA substrate binding and catalysis by the ribosome. Second, a recently developed iterative in vitro selection approach will be used to optimize the activity of ribosomes reconstituted from in vitro transcribed Bacillus stearothermophilus 23S rRNA. Finally, a variety of biochemical approaches including crosslinking, kinetic studies and chemical modification will be used to characterize the structure and function of the hybrid state of tRNA binding in translational elongation. Information obtained from these studies will eventually help to define the features of current ribosome- specific antibiotics which make them effective and the mechanism by which these same antibiotics are evaded by the host. Ultimately, this will lead to the rational design of novel antibiotics.