AbrB binding to developmentally controlled promoters

描述（由申请人提供）：枯草芽孢杆菌AbrB蛋白是一种关键的全球调节剂，可调节基因表达，以适应在次优环境下、面对压力和孢子形成发育过程的初始阶段的代谢需求。除了在快速生长过程中防止固定相相关功能的不适当表达外，有证据表明，AbrB还在调节分解代谢抑制中发挥作用，并可能影响翻译成分的生长速度调节。AbrB是一种转录调节因子，其n端结构域是一类新的DNA结合基序的范例，主要识别由不同碱基序列子集假设的微妙的三维DNA结构。超过40个编码多种代谢功能的操纵子，包括必需的产孢基因和负责产生抗菌化合物的基因，已知具有AbrB结合位点，通常位于启动子区域。在芽孢杆菌、梭状芽胞杆菌、李斯特菌和羧热菌（包括致病种）中存在的至少25种其他调节蛋白显示出广泛的氨基酸一致性和与AbrB dna结合域的同源性。对灵活的AbrB结合特异性负责的因子的阐明将提供蛋白质- dna识别机制的见解，以及细胞如何经济地使用单一蛋白质来协调各种应激反应和发育选择。检查特定残基和区域在蛋白质中所起的作用，并比较和对比由AbrB同源物序列变化指定的特性，将为具有这种灵活结合基序的蛋白质利用的大分子相互作用提供重要见解，以实现广泛但特定的DNA识别特性。abb结构与dna结合特性的关系，以及存在于结合表面和结合表面附近的特定氨基酸残基所起的确切作用，将通过突变体选择、突变体分析和从选择的AbrB同源物中检查结合域的组合来探索。假设这些蛋白质的羧基末端结构域主要是高阶多聚结构域，将使用各种方法进行测试，包括突变蛋白的遗传，生化和生物物理分析，以及具有完整或截断的c结构域融合到其他AbrB同源物的dna结合结构域的杂交蛋白的构建。抑制因子。通过这些调查获得的信息将是朝着利用AbrB基序的独特特性来设计或直接选择特异性结合任何所需DNA靶标的特定变体的最终目标迈出的关键一步。

DESCRIPTION (provided by applicant): The Bacillus subtilis AbrB protein is a key global regulator that adjusts gene expression to fit metabolic needs in suboptimal environments, in the face of stress and during the initial stages of the developmental process of sporulation. In addition to preventing inappropriate expression of stationary phase associated functions during rapid growth, evidence suggests that AbrB also plays roles in modulating catabolite repression and could affect growth-rate regulation of translational components. AbrB is a transcriptional regulator whose N-terminal domain is paradigmatic for a new class of DNA-binding motif that primarily recognizes subtle three-dimensional DNA structures that are assumed by a subset of varying base sequences. Over 40 operons encoding a wide array of metabolic functions, including essential sporulation genes and genes responsible for production of antimicrobial compounds, are known to have AbrB binding sites, usually in the promoter regions. At least 25 other regulatory proteins present in Bacillus, Clostridium, Listeria and Carboxydothermus species (including pathogenic species) show extensive amino acid identity and homology to the DNA-binding domain of AbrB. Elucidation of the factors responsible for flexible AbrB binding specificity will provide insights in protein-DNA recognition mechanisms and how a cell can economically use a single protein to coordinate a variety of stress responses and developmental options. Examining the roles played by specific residues and regions in the protein, and comparing and contrasting properties specified by sequence variations in AbrB homologs, will provide significant insights into macromolecular interactions that are exploited by proteins having this flexible binding motif in order to achieve broad, but specific, DNA recognition properties. The relationship of AbrB structure to DNA-binding properties, and the precise role played by specific amino acid residues present in and near the binding surface, will be probed by a combination of mutant selection, mutant analysis, and examination of the binding domains from selected AbrB homologs. The hypothesis that the carboxy-terminal domains of these proteins are primarily higher order multimerization domains will be tested using a variety of approaches including genetic, biochemical and biophysical analysis of mutant proteins and construction of hybrid proteins having either intact or truncated C-domains fused to the DNA-binding domains of other AbrB homologs, or to the ? Repressor. Information gained by these investigations will be a crucial step towards an ultimate goal of utilizing the unique properties of the AbrB motif in order to design, or directly select, specific variants that specifically bind any desired DNA target.