Conformational Dynamics of E. coli CytR Protein in Regulation of Gene Expression

在发育、分化或代谢调节过程中，需要对基因表达进行时间控制，这是生物学的基础。控制单个基因表达的主要机制发生在转录启动水平，并使用与特定DNA控制序列(或操纵子)结合的调节蛋白(转录激活因子或抑制因子)。这些操纵子位于RNA聚合酶结合和启动转录(启动子)的位置附近；DNA-蛋白质复合体调节转录起始复合体的形成效率，从而控制基因表达水平。多个转录调控因子通常参与调节特定基因的转录。表达水平取决于外部信号或效应器，以及操纵者及其相关调控蛋白在DNA上的排列。一些调节蛋白可以作用于许多不同的启动子，而另一些则是启动子特有的。这些组合机制产生了巨大的多样性，可以随着时间的推移提供对许多基因的协调控制以及对单个基因表达的微调。这个项目解决了在大肠杆菌中被称为CytR调节子的基因家族的组合控制机制。两种调节蛋白协同工作，在七个不相连的基因中产生协调和不同的调节模式。涉及的调节蛋白是全球基因调节因子C反应蛋白和细胞色素R特异性蛋白。这两种蛋白质之间的相互作用以及细胞色素T调节蛋白的7个基因启动子上各自操纵子元件的不同排列构成了这种复杂的组合调控的基础。该项目将采用多学科方法(I)确定调控蛋白质及其与DNA的复合体的结构和动力学，(Ii)探索蛋白质与DNA结合时相互作用的影响，以及(Iii)定量了解特定启动子序列介导的转录激活的动力学机制和该启动子上的特定蛋白质-蛋白质相互作用。这将在结构/功能水平上对组合基因调控的分子机制产生一个全面的看法。发展这种对细菌系统中基因调控的分子理解不仅将对我们理解细菌生理学，而且将对真核生物分化和发育的遗传控制产生广泛的影响。该项目还将为博士后、博士后和本科生水平的科学家提供结构生物学、分子生物物理学和分子遗传学方面的多学科培训。

The need for the temporal control of gene expression during development, differentiation or metabolic regulation is fundamental to biology. A primary mechanism for controlling expression of individual genes occurs at the level of initiation of transcription and employs regulatory proteins (transcriptional activators or repressors) that bind to specific DNA control sequences (or operators). These operators are located near the sites where RNA polymerase binds and initiates transcription (promoters); the DNA-protein complexes modulate the efficiency of formation of transcription initiation complexes and thereby control the level of gene expression. Multiple transcriptional regulators are typically involved in regulating the transcription of a particular gene. The level of expression depends on external signals or effectors, and the arrangement of the operators and their associated regulatory proteins on the DNA. Some of the regulatory proteins can act at many different promoters whereas others are promoter specific. These combinatorial mechanisms generate great diversity and can provide over time, coordinate control of many genes as well as fine-tuning of the expression of individual genes. This project addresses the combinatorial control mechanism in a gene family known as the CytR regulon in the bacterium Escherichia coli. Two regulatory proteins working in concert produce both coordinate and differential regulation patterns in seven unlinked genes. The regulatory proteins involved are the global gene regulator CRP and a CytR-specific protein. The interplay between these two proteins and the different arrangements of the respective operator elements at the seven gene promoters of the Cyt regulon underlies this complex combinatorial regulation. This project will employ a multi-disciplinary approach (i) to determine the structures and dynamics of the regulatory proteins and their complexes with DNA, (ii) to probe the effects of interactions between the proteins when bound to DNA and (iii) to develop quantitative understanding of the kinetic mechanisms of transcriptional activation mediated by the specific promoter sequence and the specific protein-protein interactions at that promoter. This will generate a comprehensive view of the molecular mechanisms of combinatorial gene regulation at the structure/function level. Developing this molecular understanding of gene regulation in this bacterial system will have broad impacts on our understanding of not only bacterial physiology, but also on the genetic control of differentiation and development in eukaryotic organisms. The project will also provide multi-disciplinary training for postdoctoral, predoctoral and undergraduate level scientists in structural biology, molecular biophysics, and molecular genetics.