Time-Resolved Hydroxyl Radical Footprinting of RNA

新核糖体的合成对于细胞生长是必需的，并且在对细胞生长的反应中受到严格的调控。 营养和压力。核糖体合成缺陷与癌症风险增加有关， 在快速生长的组织如血液和皮肤中的各种先天性缺陷。当RNA和 核糖体中的蛋白质成分相互组装形成大小 在核糖体的亚基中，局部蛋白质-RNA相互作用驱动程序化构象 RNA的变化改变了螺旋运动的方向和频率。这些蛋白质引导的 RNA动力学的变化重新排列了组装中间体的相对自由能， 控制其他蛋白质向复合物的分级组装。拟议研究 将使用单分子荧光和细胞内RNA结构探测来研究 构象开关协调30 S核糖体的各个结构域的组装，并且 每个结构域的组装如何与活性位点的折叠联系起来，活性位点位于它们的 路口单分子拉下实验和新生血管内皮细胞的体内足迹 转录本将可视化组装因子如何增加保真度和协同性， 在前rRNA转录期间组装。这项研究的成果将增加 了解细胞生长所需的大RNA-蛋白质复合物是如何组装的， 牢房它也将有助于从分子上解释人类疾病所产生的缺陷 在这些复合物的组装中。

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. As the RNA and protein components of the ribosome assemble with each other to form the large and small subunits of the ribosome, local protein-RNA interactions drive programmed conformational changes in the RNA alter the direction and frequency of helix motions. These protein-guided changes in the RNA dynamics reorder the relative free energies of assembly intermediates and control the hierarchical assembly of additional proteins to the complex. The proposed research will use single molecule fluorescence and in-cell RNA structure probing to investigate how conformational switches coordinate assembly of individual domains of the 30S ribosome, and how assembly of each domain is linked to folding of the active site, which lies at their intersection. Single-molecule pull-down experiments and in vivo footprinting of nascent transcripts will visualize how assembly factors increase the fidelity and cooperativity of assembly during transcription of the pre-rRNA. The results of this research will increase understanding of how large RNA-protein complexes necessary for cell growth are assembled in the cell. It will also contribute to a molecular explanation of human disorders arising from defects in the assembly of these complexes.