Visualizing Transcription-Coupled 30S Ribosome Assembly using Single-Molecule and Time-Resolved X-ray Footprinting

项目摘要 细胞生长是由细胞产生新的核糖体的能力驱动的，其中， 大部分的细胞转录都致力于转录核糖体RNA （rRNA）。癌细胞通常依赖于核糖体rRNA的异常表达， 增加核糖体组装速率以刺激细胞的快速生长。 核糖体组装开始于rRNA的转录过程， 过程被认为是共同调节的。然而， 转录和核糖体组装目前尚不清楚，例如 rRNA折叠或核糖体蛋白的组成存在于 转录。此外，还不知道转录过程如何影响 rRNA折叠和R-蛋白质缔合。拟议的工作旨在开发一个在 体外转录偶联核糖体组装系统，以表征 在转录过程中，16 SrRNA折叠和30 S亚基组装是转录调控的关键。 rRNA折叠景观将使用X射线和 足迹法和DMS结构探测以提供rRNA结构图 在转录方面的变化。包含核糖体蛋白和 组装因子将阐明蛋白质缔合对蛋白质组装的影响。 共转录折叠途径。这些实验将得到补充， 单分子荧光实验，将表征动力学行为 单个核糖体蛋白与新生rRNA复合物结合的过程。的 所提出的工作将提供一种新的系统，用于重现体内核糖体 组装并产生对转录偶联的基本见解 流程.一般来说，这些方法的发展将适用于 研究不同的RNP，如snoRNP或前mRNA剪接机制， 以及研究癌症中共转录过程如何失调。

Project Summary Cell growth is driven by the cell's ability to produce new ribosomes, in which the majority of cellular transcription is devoted to transcribing the ribosomal RNA (rRNA). Cancer cells often rely on aberrant expression of the ribosomal rRNA to increase rates of ribosome assembly in order to stimulate rampant cell growth. Ribosome assembly begins during transcription of the rRNA and the two processes are thought to be co-regulated. However, the relationship between transcription and ribosome assembly is currently unclear, such as the extent of rRNA folding or the composition of ribosomal proteins present during transcription. Moreover, it is unknown how the process of transcription influences rRNA folding and r-protein association. The proposed work aims to develop an in vitro transcription-coupled ribosome assembly system to characterize the effect of transcription on the process of 16S rRNA folding and 30S subunit assembly. The rRNA folding landscape will be examined using a combination of X-ray footprinting and DMS structure probing to provide a map of rRNA structural changes with respect to transcription. Inclusion of ribosomal proteins and assembly factors will elucidate the influence of protein association on the cotranscriptional folding pathway. These experiments will be complemented by single-molecule fluorescence experiments that will characterize kinetic behaviors of individual ribosomal proteins binding to nascent rRNA complexes. The proposed work will provide a novel system for recapitulating in vivo ribosome assembly and generate fundamental insights into transcription-coupled processes. In general, development of these methods will be adaptable for studying different RNPs, such as the snoRNPs or pre-mRNA splicing machinery, as well as studying how cotranscriptional processes are dysregulated in cancer.