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Global Regulatory Networks in Escherichia Coli

大肠杆菌的全球监管网络

基本信息

批准号：
7117661
负责人：
G. WESLEY HATFIELD
金额：
$ 32.69万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-19 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7117661
关键词：
DNA topoisomerases Escherichia coli bacterial genetics bacterial proteins binding sites biophysics chromosomes gene expression genetic regulation genome mathematics microarray technology molecular biology information system nucleic acid purification nucleic acid sequence nucleic acid structure operon regulatory gene

项目摘要

DESCRIPTION (provided by applicant): We have previously described DNA supercoiling-dependent mechanisms involving protein (IHF or FIS)- mediated translocation of local superhelical energy from one supercoiling-induced duplex destabilized (SIDD) region on the chromosome to another that serve to coordinate the basal levels of expression of the itvGMEDA, leuV, and ilvYC operons of the ilv regulon of Escherichia coli, both with one another and with the nutritional and environmental states of the cell. Here we propose to employ computational and biological methods to determine the extent to which these mechanisms are used for the global regulation of gene expression in this model organism. More specifically, we will use methods involving computational prediction and experimental verification to bring together three separate lines of inquiry to determine all the genes of E. coli that have the catenation of properties needed for IHF-mediated regulation by a mechanism involving a binding-induced transmission of destabilization. First, we will use computational methods to predict the locations of all the SIDD sites on the E. coli chromosome at superhelical densities encountered in otherwise isogenic wild-type, topoisomerase (topA, or gyrB) deficient E. coli strains. Second, we will develop and apply a novel method to computationally search the E. coli genome to identify all high affinity IHF binding sites. Third, we will use DNA microarrays to obtain gene expression profiles in IHF + and IHF- cells at the superhelical densities encountered in these strains. These data will allow us to identify genes (operons) that are regulated either by DNA supercoiling, or by IHF, or both. Genes whose upstream flanks contain a SIDD site that coincides with or overlaps a strong IHF binding site, and which are shown to have IHF dependent expression will be identified, and subjected to further experimental study to verify: l) that superhelicity destabilizes the predicted SIDD site; 2) that IHF binds at the predicted location; 3) that changes in duplex destabilization patterns alone, without changes in the local base sequence, affect gene regulation; and 4) that IHF binding regulates this expression in a DNA-supercoiling-dependent manner in an in vitro system.

描述(由申请人提供)：我们之前已经描述了DNA超螺旋依赖的机制，涉及蛋白质(IHF或FIS)介导的局部超螺旋能量从染色体上一个超螺旋诱导的双链不稳定(SIDD)区域转移到另一个区域，这有助于协调大肠杆菌ILV调节子的itvGMEDA、leuV和ilvYC操纵子的基础表达水平，包括相互之间以及与细胞的营养和环境状态。在这里，我们建议使用计算和生物学方法来确定这些机制在多大程度上被用于在这个模式生物中对基因表达的全球调控。更具体地说，我们将使用计算预测和实验验证的方法，将三条不同的查询路线结合在一起，以确定具有IHF介导的调节所需的特性连锁的所有大肠杆菌基因，该调节机制涉及结合诱导的失稳传递。首先，我们将使用计算方法来预测在其他同基因野生型拓扑异构酶(topA或gyrB)缺乏的大肠杆菌菌株中遇到的超螺旋密度下的所有SIDD位点的位置。其次，我们将开发和应用一种新的方法来计算搜索大肠杆菌基因组，以确定所有高亲和力IHF结合位点。第三，我们将使用DNA微阵列获得IHF+和IHF-细胞在这些菌株中遇到的超螺旋密度下的基因表达谱。这些数据将使我们能够识别受DNA超螺旋或IHF调控的基因(操纵子)，或者两者兼而有之。其上游侧翼含有与IHF结合位点重合或重叠的SIDD位点，并被证明具有IHF依赖表达的基因将被鉴定，并接受进一步的实验研究以验证：L)超螺旋使预测的SIDD位点不稳定；2)IHF在预测的位置结合；3)双链不稳定模式的变化仅影响基因调控，而不改变局部碱基序列；4)在体外系统中，IHF结合以DNA超螺旋依赖的方式调节该表达。