The Structural Dynamics of Translation Initiation

翻译起始的结构动力学

• 批准号: 9099859
• 负责人: Ruben L Gonzalez
• 金额: $ 31.88万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099859
• 关键词: Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Bacterial Proteins; Binding; Binding Sites; Biochemical; Biological; Cell physiology; Codon Nucleotides; Complex; Coupled; DNA; Development; Drug Design; Drug Targeting; Dyes; Eukaryota; Exhibits; Fluorescence; Fluorescence Microscopy; Funding; Genes; Genome; Genome engineering; Goals; Health; Human; Individual; Infection; Initiator Codon; Kinetics; Knowledge; Label; Ligands; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Multi-Drug Resistance; Organism; Pathway interactions; Peptide Initiation Factors; Phase; Play; Positioning Attribute; Process; Protein Biosynthesis; Proteins; RNA Binding; Reaction; Recruitment Activity; Research Methodology; Resistance; Ribosomes; Role; Signal Transduction; Site; Staging; Superbug; Techniques; Testing; Transfer RNA; Translating; Translation Initiation; Translations; United States; base; clinically relevant; design; mutant; next generation; novel; pathogen; programs; research study; resistant strain; single molecule; single-molecule FRET; small molecule

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.

描述（由申请人提供）：即使对目前可用的最强大的抗生素也具有抗性的细菌“超级细菌”的出现对人类健康构成严重威胁，并要求开发新的抗生素。大约一半的临床使用的抗生素靶向细菌中的蛋白质合成过程。蛋白质合成是生物体DNA基因组编码的基因的信使RNA（mRNA）拷贝通过细胞翻译机器（TM）翻译成其相应的蛋白质产物的基本细胞过程。特异性靶向细菌蛋白质合成的抗生素通过利用细菌和真核细胞TM之间的细微差异来实现。该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，细菌和真核生物最不同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，该过程始于30 S起始复合物（30 S IC）的形成，该复合物由小的30 S核糖体亚基、待翻译的mRNA、起始剂甲酰基甲硫氨酰转移RNA（tRNA）和三个起始因子（IF）组成。随后，大的50 S核糖体亚基与30 S IC结合形成功能性70 S起始复合物。亚基连接是该过程的基本特征，因此亚基连接的细菌特异性方面代表了可行的抗生素靶标。在本研究中，我们将结合分子生物学、单分子生物物理学、生物化学和结构学的策略来研究亚基连接反应的三个知之甚少的方面：在目标1中，我们将研究30 S IC结合的IFs的构象动力学如何驱动和调节亚基连接;在目的2中，我们将研究30 S亚基的结构重排和IF-和tRNA配体在30 S IC内的位置的相关变化如何调节亚基连接;在目的3中，我们将研究50 S亚基的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。基于对翻译起始的广泛的整体研究和对翻译延伸阶段的累积单分子研究，我们的指导假设是， IFs、tRNA、30 S亚基和50 S亚基随机地在各种构象状态之间波动，其中一些构象状态有利于亚基连接，而另一些构象状态是抑制性的。在该模型中，向亚基连接-活性状态的转变将上调蛋白质合成，而向亚基连接-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使活性状态不稳定或稳定抑制状态的小分子药物可以提供产生新抗生素的方法。拟议的研究将提供对亚基连接反应的全面机制理解，并且在这样做的过程中，将有助于鉴定可以作为下一代抗生素开发靶点的反应的新细菌特异性方面。