The Structural Dynamics of Translation Initiation

描述(申请人提供)：出现对目前最强大的抗生素也具有抗药性的细菌“超级细菌”，对人类健康构成严重威胁，并要求开发新的抗生素。在所有临床使用的抗生素中，大约有一半针对细菌中的蛋白质合成过程。蛋白质合成S生物体基因组编码基因的信使核糖核糖核酸拷贝被细胞翻译机器(TM)翻译成相应的蛋白质产物的基本细胞过程。专门针对细菌蛋白质合成的抗生素是通过利用细菌和真核TM之间的细微差异来做到这一点的。这项建议的最终目标是通过在细菌TM中识别新的抗生素靶点来为抗生素开发工作提供信息。在这里，我们专注于翻译的起始阶段，这是细菌和真核生物之间差异最大的蛋白质合成途径的步骤。翻译起始是一个动态的、多步骤的过程，在细菌中，首先形成一个30S起始复合体(30S IC)，它由小的30S核糖体亚基、待翻译的mRNA、启动子甲硫酰转移RNA(TRNA)和三个起始因子(IF)组成。随后，大的50S核糖体亚基与30S IC连接，形成功能性的70S起始复合体。亚基连接是这一过程的一个基本特征，因此，亚基连接的细菌特异性方面代表了可行的抗生素靶点。在这个方案中，我们将结合分子生物学、单分子生物物理、生化和结构策略来研究亚基连接反应的三个鲜为人知的方面：在目标1中，我们将研究30S IC结合的IF的构象动力学如何驱动和调节亚单位连接；在目标2中，我们将研究30S亚基的结构重排以及相关的IF-和tRNA配体在30S IC内的位置变化如何调节亚基连接；在目标3中，我们将研究50S亚基的因子结合位点的单个成分在指导亚基连接反应中的作用。我们的指导性假设是，基于广泛的翻译启动研究和积累的关于翻译延伸阶段的单分子研究，我们的指导假设是 IFs、tRNA、30S亚基和50S亚基在各种构象状态之间随机波动，其中一些有助于亚基连接，另一些则是抑制。在这个模型中，向亚单位连接-能力状态的转变将上调蛋白质合成，而向亚单位连接-抑制状态的转变将下调蛋白质合成；因此，开发旨在以细菌特异性方式破坏竞争状态或稳定抑制状态的小分子药物可以提供一种产生新抗生素的方法。拟议的研究将提供对亚单位加入反应的全面机制理解，并通过这样做，将有助于确定新的细菌特有的反应方面，这些方面可以作为下一代抗生素开发的目标。

DESCRIPTION (provided by applicant): The emergence of bacterial "superbugs" with resistance to even the most powerful antibiotics currently available poses a serious threat to human health and demands the development of new antibiotics. Approximately half of all clinically used antibiotics target the process of protein synthesis in bacteria. Protein synthesis s an essential cellular process whereby messenger RNA (mRNA) copies of the genes encoded by an organism's DNA genome are translated into their corresponding protein products by the cellular translational machinery (TM). Antibiotics that specifically target bacterial protein synthesis do so by exploiting subtle differences between the bacterial and eukaryotic TMs. The ultimate goal of this proposal is to inform antibiotic development efforts through the identificatin of new antibiotic targets within the bacterial TM. Herein, we focus on the initiation phase of translation, the step of the protein synthesis pathway where bacteria and eukaryotes differ the most. Translation initiation is a dynamic, multi-step process that, in bacteria, begins with the formation of a 30S initiation complex (30S IC) comprised of the small 30S ribosomal subunit, the mRNA to be translated, an initiator formylmethionyl-transfer RNA (tRNA), and three initiation factors (IFs). Subsequently, the large 50S ribosomal subunit joins to the 30S IC to form a functional 70S initiation complex. Subunit joining is an essential feature of this process and bacterial-specific aspects of subunit joining consequently represent viable antibiotic targets. In this proposal, we will use a combination of molecular biological, single-molecule biophysical, biochemical, and structural strategies to investigate three poorly understood aspects of the subunit joining reaction: In Aim 1, we will investigate how the conformational dynamics of the 30S IC-bound IFs drive and regulate subunit joining; In Aim 2, we will examine how structural rearrangements of the 30S subunit and associated changes in the positions of IF- and tRNA ligands within the 30S IC regulate subunit joining; In Aim 3, we will study the roles that the individual components of the factor-binding site of the 50S subunit play in directing the subunit joining reaction. Our guiding hypothesis, based on extensive ensemble studies of translation initiation and accumulating single-molecule studies of the elongation phase of translation, is that the IFs, tRNA, 30S subunit, and 50S subunit stochastically fluctuate between various conformational states, some of which are conducive to subunit joining, and others that are inhibitory. In this model, shifts towards subunit joining-competent states would up-regulate protein synthesis, while shifts towards subunit joining-inhibitory states would down-regulate protein synthesis; development of small-molecule drugs designed to destabilize the competent states or stabilize the inhibitory states in a bacteria-specific manner could therefore provide a means of generating new antibiotics. The proposed studies will provide a comprehensive mechanistic understanding of the subunit joining reaction and, in doing so, will aid in the identification of novel bacteria- specific aspects of the reaction that can serve as targets for th development of next-generation antibiotics.