The Structural Dynamics of Translation Initiation

描述（由申请人提供）：细菌“超级细菌”的出现对目前可用的最强大的抗生素产生耐药性，对人类健康构成严重威胁，要求开发新的抗生素。在所有临床使用的抗生素中，大约有一半是针对细菌中蛋白质合成过程的。蛋白质合成是一个重要的细胞过程，通过细胞翻译机制（TM）将生物体DNA基因组编码的基因的信使RNA （mRNA）拷贝翻译成相应的蛋白质产物。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生物TMs之间的细微差异来实现的。本提案的最终目标是通过鉴定细菌TM中的新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区别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过程，在细菌中，从30S起始复合物（30S IC）的形成开始，该复合物由30S核糖体小亚基、待翻译mRNA、启动物甲酰基甲硫基转移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结合，形成功能性的70S起始复合物。亚基连接是这一过程的基本特征，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。在本研究中，我们将结合分子生物学、单分子生物物理学、生物化学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Aim 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基连接；在Aim 2中，我们将研究30S亚基的结构重排以及30S IC中IF-和tRNA配体位置的相关变化如何调节亚基连接；在Aim 3中，我们将研究50S亚基因子结合位点的各个组分在指导亚基连接反应中所起的作用。我们的指导假设，基于广泛的翻译起始的综合研究和积累的翻译延伸期的单分子研究，是

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.