Time-resolved hydroxyl radical footprinting of RNA

描述(由申请人提供)：新核糖体的合成对细胞生长至关重要，并受到严格调控，以应对营养和压力。核糖体合成缺陷与癌症风险增加以及血液和皮肤等快速生长组织中的各种先天性缺陷有关。这项研究的目的是了解核糖体的RNA和蛋白质组分如何相互组装形成核糖体的大小亚基，并了解这一过程是如何被细胞中的其他蛋白质加速和调节的。时间分辨足迹可以在10-20毫秒内可视化RNA和蛋白质相互作用的变化，从而获得关于折叠的详细信息 核糖核酸和核糖核酸-蛋白质识别途径。该方法将用于研究30S亚基头部RNA-蛋白质复合体的缓慢重塑，并研究成熟因子如何提高蛋白质结合和组装的速度。同步加速器X射线束将被用来在活细菌中追踪30S组装中间体的足迹。利用单个RNA-蛋白质复合体的荧光光谱可以直接观察到rRNA中构象开关的动力学。这项研究的结果将增加对细胞生长所需的大RNA-蛋白质复合体的理解，并有助于从分子上解释由这些复合体的组装缺陷引起的人类疾病。

DESCRIPTION (provided by applicant): The synthesis of new ribosomes is essential for cell growth, and tightly regulated in response to nutrients and stress. Defects in ribosome synthesis are linked to increased risk of cancer and a variety of congenital defects in rapidly growing tissues such as blood and skin. The aim of this research is to understand how the RNA and protein components of the ribosome assemble with each other to form the large and small subunits of the ribosome, and to understand how this process is accelerated and regulated by other proteins in the cell. Time-resolved footprinting can visualize changes in the RNA and protein interactions within 10-20 milliseconds, resulting in detailed information about the folding pathway of the RNA and RNA-protein recognition. This method will be used to investigate slow remodeling of RNA-protein complexes in the head of the 30S subunit, and study how maturation factors increase the rate of protein binding and assembly. A synchrotron X-ray beam will be used to footprint 30S assembly intermediates in living bacteria. The dynamics of a conformational switch in the rRNA will be observed directly using fluorescence spectroscopy of single RNA-protein complexes. The results of this research will increase understanding of how large RNA-protein complexes are necessary for cell growth, and contribute to a molecular explanation of human disorders arising from defects in the assembly of these complexes.