Eukaryotic Translational Initiation and its Regulation

描述（由申请人提供）：蛋白质合成的起始受到高度调控，是细胞信号通路的终点；起始的功能失调与癌症密切相关。起始涉及许多宿主因子，这些因子协助核糖体亚基结合到mrna的5'端；随后，这个核糖体复合体定向扫描到第一个起始密码子，在那里一个80S复合体组装，准备开始蛋白质合成。尽管对这些成分的结构知识越来越多，但对翻译起始及其调控的完整机制和动态理解仍然难以捉摸。在这里，我们利用生化方法将单分子荧光方法应用于真核生物翻译起始。在具体目标1中，将使用单分子荧光来确定启动因子和40S亚基结合到模型mRNA的时间和顺序，从而无需扫描即可启动。在与j.l orsch （JHU）的合作中，我们将使用染料标记的酵母翻译因子。在具体的目标2中，我们将研究起始的后期步骤，如GTP水解和60S亚基连接。我们将确定亚基连接的时间和动力学以及GTP水解在这些步骤中的作用。我们将使用抗生素和毒素来描述起始的机制步骤。在具体目标3中，我们将通过在真实的mRNA 5'UTR上添加5'UTR扫描来进一步构建系统的复杂性。我们将观察核糖体亚基和mRNA之间的FRET，以监测起始复合物到起始密码子的运动。我们将测试5'UTR中mRNA结构对扫描动力学的作用。在具体目标4中，我们将探讨所提出的5‘到3’端圆化在调制起始效率中的性质和持续时间。我们将通过检测翻译核糖体上的染料标记蛋白，将这些观察结果与蛋白质生产的观察结果结合起来。本提案的结果将为理解真核翻译、其调控和在人类疾病中的作用提供生物物理背景。

DESCRIPTION (provided by applicant): Initiation of protein synthesis is highly regulated and is the endpoint of cellular signaling pathways; misfunction of initiation is strongly linked to cancer. Initiation involves many host factors that assist ribosomal subunit binding to the 5'end of mRNAs; subsequently, this ribosomal complex directionally scans to the first start codon, where an 80S complex assembles, poised to start protein synthesis. Despite increasing structural knowledge of the components, a full mechanistic and dynamic understanding of translation initiation and its regulation remains elusive. Here, we harness biochemical approaches to apply single-molecule fluorescence methods to eukaryotic translation initiation. In specific aim 1, single-molecule fluorescence will be used to determine the timing and order of initiation factor and 40S subunit binding to a model mRNA that allows initiation without scanning. In collaboration with J. Lorsch (JHU), we will use dye-labeled yeast translation factors. In specific aim 2, we will study later steps in initiation, such as GTP hydrolysis and 60S subunit joining. We will determine the timing and dynamics of subunit joining and the role of GTP hydrolysis in these steps. We will use antibiotics and toxins to delineate mechanistic steps in initiation. In specific aim 3, we will build further complexity into the system by adding 5'UTR scanning on a real mRNA 5'UTR. We will observe FRET between ribosomal subunit and mRNA to monitor movements of the initiation complex to the start codon. We will test the role of mRNA structure in the 5'UTR on scanning dynamics. In specific aim 4, we will probe the nature and duration of proposed 5'- to 3'- end circularization in modulating initiation efficiency. We will couple these observations to those of protein production through detection of dye-labeled protein on the translating ribosome. The results of this proposal will provide a biophysical context to understanding eukaryotic translation, its regulation, and role in human disease.