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

描述(申请人提供)：转录控制是通过转录因子电池与DNA相互作用，以及与转录装置本身相互作用。在许多情况下，结合到远离基础转录装置的数百到数千个碱基对的调控区域的蛋白质通过同时与DNA上的一个以上位置结合来执行一种距离较远的生物作用，导致基因组DNA的大规模重排(例如DNA环)。然而，转录调控的机制基础，特别是涉及DNA大规模变形的转录调控的机制基础，以及这种调控的定量结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互关联的支柱上来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和基因表达水平之间的关系，ii)使用单分子拴系粒子实验来测量转录因子-DNA复合体形成的稳定性和动力学，以及iii)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)、结合位点强度和操纵子数量都被用作调节基因表达水平的刻度盘。与公共卫生相关：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。作为研究基因及其调控方式的中心线索之一，转录控制在生物学和医学上都是相关的。这里提出的工作旨在以一种将从合成生物学应用到病原体研究的方式来加强我们对微生物转录的理解。

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.