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

使用单分子和时间分辨 X 射线足迹可视化转录偶联的 30S 核糖体组装

• 批准号: 9815918
• 负责人: Margaret Louise Rodgers
• 金额: $ 6.16万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2020-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9815918
• 关键词: Active Sites; Address; Behavior; Binding; Binding Proteins; Binding Sites; Biological Assay; Cell Survival; Cells; Complement; Complex; Coupled; DNA-Directed RNA Polymerase; Data; Defect; Disease; Fluorescence; Genes; Genetic Transcription; Heterogeneity; Hydroxyl Radical; In Vitro; Individual; Kinetics; Lead; Length; Malignant Neoplasms; Maps; Measures; Methods; Modeling; Molecular Conformation; Molecular Machines; Monitor; Mutation; Normal Cell; Organism; Pathway interactions; Pattern; Process; Property; Proteins; RNA; RNA Folding; RNA Splicing; RNA, Ribosomal, 16S; Regulation; Reporting; Research; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Roentgen Rays; Sampling; Small Nucleolar Ribonucleoproteins; Speed; Spliceosomes; Structure; System; Testing; Time; Transcription Process; Work; cancer cell; cell growth; cost; design; experimental study; human disease; in vivo; insight; mRNA Precursor; method development; mutant; novel; overexpression; single molecule; time use; vector

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.

项目概要 细胞生长是由细胞产生新核糖体的能力驱动的，其中 大部分细胞转录致力于转录核糖体 RNA （rRNA）。癌细胞通常依赖于核糖体 rRNA 的异常表达 提高核糖体组装速度，以刺激细胞的疯狂生长。 核糖体组装在 rRNA 转录过程中开始，并且两者 过程被认为是共同监管的。然而，之间的关系 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 转录。此外，尚不清楚转录过程如何影响 rRNA 折叠和 r 蛋白关联。拟议的工作旨在开发一种 体外转录偶联核糖体组装系统来表征效果 16S rRNA 折叠和 30S 亚基组装过程中转录的影响。 rRNA 折叠景观将使用 X 射线组合进行检查 足迹分析和 DMS 结构探测提供 rRNA 结构图 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关联对 共转录折叠途径。这些实验将得到补充 表征动力学行为的单分子荧光实验 单个核糖体蛋白与新生 rRNA 复合物的结合。这 拟议的工作将为重现体内核糖体提供一种新的系统 组装并生成对转录耦合的基本见解 流程。一般来说，这些方法的开发将适用于 研究不同的 RNP，例如 snoRNP 或前 mRNA 剪接机制， 以及研究癌症中共转录过程如何失调。