RUI:Transcriptional Activation by the Bacillus Subtilis ComK Protein

本研究的目的是阐明枯草芽孢杆菌能力转录因子ComK激活转录的机制。枯草芽孢杆菌的能力以细胞结合和吸收外源DNA的能力为特征。除了控制自身的表达外，ComK对于组成DNA结合和摄取系统的蛋白质的表达以及重组基因recA和addAB的能力相关表达至关重要。ComK结合在小槽序列上游的-35区域，并导致弯曲的DNA。根据ComK二聚体结合位点之间的间距将ComK调控启动子分为三类：第一类（recA、addAB、dinA和nucA）二聚体结合位点之间的间距为2个螺旋匝；第二类，包括晚期能力基因（comC, -G， -E和-F），包含3个螺旋旋转的结合位点；第三类包含一个单一的代表，即comK启动子，其结合位点被4个螺旋旋转分开。所有comk依赖性启动子的下游结合位点都位于大肠杆菌RNA聚合酶α亚基结合的区域，该区域也结合在次要凹槽中。PI将表征ComK的DNA结合域，并验证ComK影响RNA聚合酶a亚基结合的假设。ComK DNA结合域将使用突变分析、核磁共振结构测定和分子建模相结合的方法进行表征。PI将通过结合羟基自由基足迹、靶向足迹、蛋白质蛋白质交联和突变分析，确定当ComK与三种ComK依赖性启动子recA、comG和ComK的代表结合时，ComK是否与RNA聚合酶α相互作用。我们将通过迁移转移试验、体外转录试验以及recA-lacZ、comG-lacZ和ComK - lacz融合体的转录激活分析来评估野生型和突变型ComK蛋白的DNA结合和转录激活。这项研究包括调查ComK蛋白如何开启一系列基因的表达，这些基因使枯草芽孢杆菌能够吸收外部DNA并将这些DNA整合到其染色体中。这些结果将阐明ComK如何控制枯草芽孢杆菌细胞进入特殊状态进行基因交换时所需的数十个基因的表达。由于ComK的作用可能涉及与RNA聚合酶的相互作用，RNA聚合酶是控制所有细菌基因表达的酶，因此本研究应进一步了解细菌基因的一般调控。

The objective of this research is to elucidate the mechanism by which the Bacillus subtilis competence transcription factor, ComK, activates transcription. Competence in B. subtilis is characterized by the cell's ability to bind and take up exogenous DNA. In addition to controlling its own expression, ComK is essential for the expression of the proteins that comprise the DNA binding and uptake system and for the competence-related expression of the recombination genes recA and addAB. ComK binds in the minor groove to sequences upstream of the -35 region and causes bending of the DNA. Three classes of ComK-regulated promoters are distinguished based on the spacing between ComK dimer binding sites: In the first class (recA, addAB, dinA, and nucA) the interval between dimer binding sites is 2 helical turns; the second class, comprising the late competence genes (comC, -G, -E, and -F), contain binding sites separated by 3 helical turns; and the third class contains a single representative, the comK promoter, in which the binding sites are separated by 4 helical turns. The downstream binding sites for all ComK-dependent promoters are located in regions proposed for the binding of the alpha subunits of E. coli RNA polymerase, which also binds in the minor groove. The PI will characterize the DNA binding domain of ComK and test the hypothesis that ComK affects the binding of the a subunits of RNA polymerase. The ComK DNA binding domain will be characterized using a combination of mutational analysis, NMR structural determination, and molecular modeling. The PI will determine whether or not ComK interacts with RNA polymerase alpha when it binds to representatives of the three classes of ComK-dependent promoters, recA, comG, and comK, by a combination of hydroxyl radical footprinting, targeted footprinting, protein-protein crosslinking, and mutational analyses. DNA binding and transcriptional activation will be assessed for wild-type and mutant ComK proteins using mobility shift assays, in vitro transcription assays, and analyses of transcriptional activation of recA-lacZ, comG-lacZ, and comK-lacZ fusions.This research involves investigating the way in which the ComK protein turns on the expression of an assortment of genes that enable the bacterium Bacillus subtilis to take up external DNA and incorporate this DNA into its chromosome. The results should elucidate how ComK controls the expression of dozens of genes that are required when Bacillus subtilis cells enter a specialized state for genetic exchange. Since the action of ComK likely involves interactions with RNA polymerase, the enzyme that governs gene expression in all bacteria, this research should further the understanding of bacterial gene regulation in general.