Single-Molecule and Single-Cell Analysis of Transcription Factor-DNA Complexes

转录因子-DNA 复合物的单分子和单细胞分析

• 批准号: 7771677
• 负责人: ROB PHILLIPS
• 金额: $ 34.08万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-17 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7771677
• 关键词: Amino Acid Sequence; Architecture; Area; Award; Binding; Binding Sites; Biological; Biology; Case Study; Cell Differentiation process; Cells; Characteristics; Chromatin Loop; Collection; Complex; Cytometry; DNA; DNA Sequence; DNA Sequence Rearrangement; Data; Dependence; Eukaryota; Evolution; Exhibits; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Grant; Heart; Kinetics; Lac Operon; Length; Life; Life Cycle Stages; Location; Measurement; Measures; Mediating; Medicine; Microbe; Microbial Biofilms; Microscopy; Modeling; Nucleic Acid Regulatory Sequences; Nucleosomes; Organism; Pattern; Peptide Sequence Determination; Pilot Projects; Plasmids; Positioning Attribute; Probability; Process; Prokaryotic Cells; Protein Binding; Proteins; Repressor Proteins; Research; Role; Site; Statistical Mechanics; System; Techniques; Testing; Thermodynamics; Time; Transcriptional Regulation; Tweens; Work; flexibility; particle; pathogen; promoter; public health relevance; research study; response; single cell analysis; single molecule; synthetic biology; theories; transcription factor

DESCRIPTION (provided by applicant): Transcriptional control is mediated by batteries of transcription factors that interact with DNA, each other and with the transcription apparatus itself. In many of these cases, proteins bound to regulatory regions hundreds to thousands of basepairs away from the basal transcription apparatus perform a kind of biological action at a distance by binding at more than one site on the DNA simultaneously resulting in wholesale rearrangements of the genomic DNA (e.g. DNA looping). However, the mechanistic underpinnings of transcriptional control, especially that involving large-scale deformations of the DNA, and the quantitative consequences of this control largely remain to be elucidated. The work proposed here builds upon three distinct but related pillars to analyze the formation of these complexes: i) the use of statistical mechanics models to compute the relation between regulatory architecture and the level of gene expression, ii) the use of single-molecule tethered particle experiments to measure the stability and kinetics of formation of transcription factor-DNA complexes and iii) the measurement of level of gene expression in living cells. In all three of these cases, DNA sequence (flexibility), binding site strength and number of operators are used as dials to tune the level of gene expression. PUBLIC HEALTH RELEVANCE: Transcriptional regulation is at the heart of processes in biology ranging from the formation of biofilms to cell differentiation to evolution. In its role as one of the central threads in the study of genes and how they are regulated, transcriptional control is relevant to biology and medicine alike. The work proposed here aims to strengthen our understanding of transcription in microbes in a way that will have applications from synthetic biology to the study of pathogens.

描述（由申请人提供）：转录控制由一系列转录因子介导，这些转录因子与DNA相互作用，相互作用以及与转录装置本身相互作用。在这些情况中的许多情况下，与距离基础转录装置数百至数千个碱基对的调节区结合的蛋白质通过在DNA上的一个以上位点同时结合而在一定距离处执行一种生物作用，从而导致基因组DNA的大规模重排（例如DNA成环）。然而，转录控制的机制基础，特别是涉及DNA的大规模变形，以及这种控制的定量后果在很大程度上仍有待阐明。这里提出的工作建立在三个不同但相关的支柱上，以分析这些复合体的形成：i）使用统计力学模型来计算调控结构与基因表达水平之间的关系，ii）使用单分子拴系颗粒实验来测量转录因子-DNA复合物形成的稳定性和动力学，以及iii）对活细胞中基因表达水平的测量。在所有这三种情况下，DNA序列（灵活性），结合位点的强度和操作员的数量被用作调节基因表达水平的刻度盘。公共卫生相关性：转录调控是生物学过程的核心，从生物膜的形成到细胞分化再到进化。转录调控是研究基因及其调控方式的中心线索之一，它与生物学和医学都有关。这里提出的工作旨在加强我们对微生物转录的理解，这种理解将从合成生物学应用到病原体研究。