DEFINING THE ROLE PLAYED BY 23S RRNA IN TRANSLATION

将细胞中的遗传信息翻译成功能蛋白质是一种基本的、高度保守的功能。核糖体是细菌中由三个大RNA(RRNAs)和50多个蛋白质(r-蛋白质)组成的两个亚单位的大分子复合体，是复杂而协调的翻译过程的催化剂和框架。多肽基转移酶可能是核糖体最原始的活性，是最重要的。对生命起源的理论考虑预测了rRNA在翻译中的中心作用，这一预测因RNA的催化作用而得到证实，并与在rRNA序列之间观察到的极端系统发育保守一致。广泛的生化和遗传学研究表明，rRNAs在翻译过程中发挥着主要的功能作用，特别是23S rRNA在中心催化事件--多肽键的形成中发挥着重要作用。对核糖体的结构研究，包括冷冻电子显微镜和X射线结晶学，正在产生丰富的信息。然而，这些方法所产生的核糖体的静态观点并不能揭示复杂的翻译过程的机制或动力学。这项建议描述了在体外遗传和生化方法，以确定在活性部位的分子组成的肽基转移酶(RNA或蛋白质)。这些研究的目的是了解这些成分如何协同形成多肽键，并了解如何在每个催化循环之间控制这种活性，以允许产生高保真编码的蛋白质产品。首先，将使用定点突变和修饰干扰的方法来确定23S rRNA中与核糖体与tRNA底物结合和催化有关的核苷酸。其次，一种新开发的迭代体外选择方法将用于优化由体外转录的嗜热脂肪芽孢杆菌23S rRNA重组的核糖体的活性。最后，将使用包括交联、动力学研究和化学修饰在内的各种生化方法来表征tRNA结合在翻译延伸中的杂交状态的结构和功能。从这些研究中获得的信息最终将有助于确定当前核糖体特异性抗生素的特征，使它们有效，以及这些相同的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.