Eukaryotic Translational Initiation and its Regulation

描述(申请人提供)：蛋白质合成的启动受到高度调控，是细胞信号通路的终点；启动功能障碍与癌症密切相关。起始涉及许多宿主因素，帮助核糖体亚单位与mRNAs的5‘端结合；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80s复合体组装，准备开始蛋白质合成。尽管对这些组成部分的结构知识越来越多，但对翻译启动及其调控的全面机械性和动态化理解仍然是难以捉摸的。在这里，我们利用生化方法将单分子荧光方法应用于真核细胞的翻译启动。在具体目标1中，将使用单分子荧光来确定启动因子和40S亚基与允许无需扫描的启动的模型mRNA结合的时间和顺序。在与J.Lorsch(JHU)的合作中，我们将使用染料标记的酵母翻译因子。在具体目标2中，我们将研究启动的后续步骤，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启蒙过程中的机械性步骤。在具体目标3中，我们将通过在真实的mRNA5‘非编码区上添加5’非编码区扫描来增加系统的复杂性。我们将观察核糖体亚基和mRNA之间的FRET，以监测起始复合体向起始密码子的移动。我们将测试5‘非编码区中的信使核糖核酸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‘端至3’端环化在调节引发效率方面的性质和持续时间。我们将通过检测翻译核糖体上的染料标记蛋白质来将这些观察结果与蛋白质生产相结合。这一提议的结果将为理解真核翻译、其调控及其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.