Global Regulatory Networks in Escherichia Coli

大肠杆菌的全球监管网络

• 批准号: 6677631
• 负责人: G. WESLEY HATFIELD
• 金额: $ 33.21万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677631
• 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

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）： 我们先前描述了DNA超卷曲依赖机制涉及蛋白质（IHF或FIS）介导的局部超螺旋能量从染色体上的一个超卷曲诱导的双链体不稳定（SIDD）区域到另一个区域的易位，所述区域用于协调大肠杆菌的ilv调节子的itvGMEDA、leuV和ilvYC操纵子的基础表达水平，既与彼此又与细胞的营养和环境状态。在这里，我们建议采用计算和生物学的方法来确定在何种程度上这些机制被用于在这个模式生物的基因表达的全局调节。更具体地说，我们将使用包括计算预测和实验验证的方法，将三条独立的调查路线结合在一起，以确定E.大肠杆菌，具有通过涉及结合诱导的不稳定传递的机制的IHF介导的调节所需的属性的连锁。首先，我们将使用计算方法来预测E.大肠杆菌染色体在超螺旋密度遇到，否则等基因野生型，拓扑异构酶（topA，或gyrB）缺陷的E。大肠杆菌菌株。其次，我们将开发和应用一种新的方法来计算搜索E。coli基因组中鉴定所有高亲和力IHF结合位点。第三，我们将使用DNA微阵列获得在IHF +和IHF-细胞中的基因表达谱，在这些菌株中遇到的超螺旋密度。这些数据将使我们能够识别基因（操纵子），无论是由DNA超螺旋，或IHF，或两者兼而有之。将鉴定其上游侧翼含有与强IHF结合位点重合或重叠的SIDD位点的基因，并且将其显示具有IHF依赖性表达，并进行进一步的实验研究以验证：1）超螺旋性使预测的SIDD位点不稳定; 2）IHF结合在预测的位置; 3）仅双链体不稳定模式的变化，而局部碱基序列没有变化，影响基因调控;和4）IHF结合在体外系统中以DNA超螺旋依赖性方式调控这种表达。