Time-Resolved Hydroxyl Radical Footprinting of RNA

新的核糖体的合成对细胞的生长至关重要，并受到严格的调控，以应对 营养物质和压力。核糖体合成缺陷与癌症风险增加和 血液和皮肤等生长迅速的组织中的各种先天缺陷。作为RNA和 核糖体的蛋白质成分相互组装，形成大大小小的 核糖体的亚基，局部的蛋白质-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。这些由蛋白质引导的 RNA动力学的变化重新排列了组装中间体和 控制更多蛋白质对复合体的分层组装。拟议的研究 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30S核糖体各个结构域的组装，以及 每个结构域的组装如何链接到活动站点的折叠，活动站点位于其 交叉口。新生儿的单分子下拉实验及体内足迹研究 成绩单将直观地显示组装因子如何提高 在转录前rRNA的过程中组装。这项研究的结果将会增加 理解细胞生长所需的大RNA-蛋白质复合体是如何组装在一起的 牢房。这也将有助于从分子上解释缺陷引起的人类疾病。 在这些复合体的组装中。

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.