Quantitative Characterization & Modeling of sRNA-Mediated Gene Regulation

描述（由申请人提供）：近年来，人们越来越认识到小调控RNA （small regulatory RNA, sRNA）在原核生物和真核生物中协调基因活动中所起的复杂作用。目前对细菌中srna介导的基因调控的分子组成和机制已经有了基本的了解。我们实验室的理论分析表明，sRNA介导的基因调控具有许多独特的功能特征，包括阈值线性响应和对噪声波动的抵抗，这对涉及sRNA的遗传回路的特性具有有趣的影响。在此，我们提出了一个联合实验/计算研究计划，以表征大肠杆菌中sRNA的多个尺度的功能。在分子尺度上，我们将通过表征所选突变体的基因表达，并构建相互作用的生物物理/生物信息学模型，阐明最常见的ryhb类sRNA-靶标相互作用的序列决定因素。在“装置”尺度上，我们将描述预测的sRNA介导调控的各种新特性，包括同一sRNA的不同靶点之间的分层串扰。在电路尺度上，我们将构建合成电路，研究常见电路基元（如级联和开关）的特性，并研究它们的时间特性。此外，我们将开发模型来设计针对和沉默特定宿主基因的反义srna。我们期望分子相互作用的知识能够带来预测工具，以识别潜在的大量sRNA靶标，并构建涉及大肠杆菌中sRNA的基因调控网络的额外层，而对设备和常见电路基序的了解将有助于理解sRNA调节剂在协调更大规模网络中可能发挥的特殊作用。此外，内源性靶点的沉默可能导致产生多基因敲低菌株的有效工具，可用于药物发现研究以及全基因组范围内基因-基因相互作用的研究。

DESCRIPTION (provided by applicant): In recent years, there has been a growing appreciation for the complex roles that small regulatory RNA (sRNA) can play in coordinating gene activities in both prokaryotes and eukaryotes. There exists by now a basic understanding of the molecular components and mechanisms involved in sRNA-mediated gene regulation in bacteria. Theoretical analysis by our lab suggest a number of unique functional features for sRNA-mediated gene regulation, including a threshold-linear response and a resistance to noisy fluctuation, with interesting implications on properties of genetic circuits involving sRNA. Here we propose a joint experimental/computational research program to characterize the functions of sRNA in E. coli at multiple scales. At the molecular scale, we will elucidate the sequence determinant of sRNA-target interaction for the most common RyhB-class sRNA by characterizing the gene expression of selected mutants, and constructing biopysical/bioinformatic models of the interaction. At the "device" scale, we will characterize various novel properties predicted for sRNA-mediated regulation, including hierarchical cross talk between different targets of the same sRNA. At the circuit scale, we will construct synthetic circuits and study the properties of common circuit motifs such as the cascade and switches, and investigate their temporal characteristics. In addition we will develop models to design antisense sRNAthat target and silence specific host genes. We expect that the knowledge of the molecular interaction to lead to predictive tools to identify the potentially large number of sRNA targets and construct additional layers of the gene regulatory network involving sRNA in E. coli, while knowledge of the devices and common circuit motifs will shed light towards understanding the special roles that sRNA regulators may play in coordinating larger scale networks. Additionally, the silencing of endogenous target may lead to an effective tool to generate multiple gene knockdown strains which may be used for drug discover studies as well as gene- gene interaction studies at the genome-wide scale.