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种重要且高度保守的功能。 核糖体是一种由两个亚基组成的大分子复合物，由细菌中的三个大RNA（rRNA）和50多个蛋白质（r-蛋白）组成，是复杂和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性，具有核心重要性。 对生命起源的理论思考预测了rRNA在翻译中的核心作用，这一预测因RNA催化作用的证明而得到加强，并与rRNA序列中观察到的极端系统发育保守性相一致。 广泛的生物化学和遗传学研究表明，rRNA在翻译过程中发挥主要的功能作用，特别是23S rRNA在中心催化事件，肽键形成中。 核糖体的结构研究，包括冷冻电子显微镜和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不会揭示复杂的翻译过程的机制或动力学。该提案描述了体外遗传和生物化学方法来鉴定肽基转移酶（RNA或蛋白质）活性位点的分子组分。 这些研究的目的是了解这些组分如何共同形成肽键，并了解如何在每个催化循环之间控制这种活性，以产生高保真编码的蛋白质产物。 首先，将使用定点突变和修饰干扰方法来鉴定23 S 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从体外转录的嗜热脂肪芽孢杆菌23S rRNA重建的核糖体的活性。 最后，各种生化方法，包括交联，动力学研究和化学修饰将被用来表征的结构和功能的杂交状态的tRNA结合在翻译延伸。 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，这些特征使它们有效，以及宿主逃避这些相同抗生素的机制。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.