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.

项目摘要 细胞生长是由细胞产生新核糖体的能力驱动的，在核糖体中 大部分细胞转录都是用来转录核糖体rna的。 (RRNA)。癌细胞通常依赖于核糖体rRNA的异常表达来 增加核糖体组装的速度，以刺激疯狂的细胞生长。 核糖体组装开始于rRNA和两者的转录过程 这一过程被认为是共同监管的。然而，两国之间的关系 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 抄写。此外，转录过程如何影响尚不清楚。 RRNA折叠和r-蛋白结合。拟议的工作旨在开发一个In 体外转录偶联核糖体组装系统研究其作用 转录在16S rRNA折叠和30S亚基组装过程中的作用。 将使用X射线的组合来检查rRNA折叠情况 足迹和DMS结构探测以提供rRNA结构图 与转录有关的变化。包括核糖体蛋白和 组装因子将阐明蛋白质缔合对蛋白质的影响 共转录折叠途径。这些实验将得到以下补充 将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复合体结合。这个 拟议的工作将为体内核糖体的重现提供一种新的系统 汇编并生成对转录耦合的基本见解 流程。一般而言，这些方法的发展将适用于 研究不同的RNPs，如snoRNPs或前mRNA剪接机制， 以及研究共转录过程在癌症中是如何失调的。