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局调节因子，在面临压力和孢子形成发育过程的初始阶段，调节基因表达以适应次优环境中的代谢需求。除了防止在快速生长过程中稳定期相关功能的不适当表达外，有证据表明AbrB还在调节分解代谢产物抑制中发挥作用，并可能影响翻译组分的生长速率调节。AbrB是一种转录调节因子，其N-末端结构域是一类新的DNA结合基序的典范，该基序主要识别由不同碱基序列的子集假定的微妙的三维DNA结构。已知超过40个操纵子编码广泛的代谢功能，包括必需的孢子形成基因和负责产生抗微生物化合物的基因，具有AbrB结合位点，通常在启动子区。存在于芽孢杆菌属、梭菌属、李斯特菌属和一氧化碳热菌属物种（包括致病物种）中的至少25种其他调节蛋白显示出与AbrB的DNA结合结构域的广泛氨基酸同一性和同源性。阐明负责灵活的AbrB结合特异性的因素将提供蛋白质-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。检查特定的残基和区域在蛋白质中所发挥的作用，并比较和对比AbrB同源物的序列变异所指定的特性，将提供重要的见解大分子相互作用，利用蛋白质具有这种灵活的结合基序，以实现广泛的，但具体的，DNA识别特性。AbrB结构与DNA结合特性的关系，以及存在于结合表面中和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，突变体分析和检查所选AbrB同系物的结合结构域的组合来探讨。假设这些蛋白质的羧基末端结构域主要是高阶多聚化结构域将使用多种方法进行测试，包括遗传，生物化学和生物物理分析的突变蛋白质和杂交蛋白质的构建具有完整或截短的C-结构域融合到其他AbrB同源物的DNA结合结构域，或？镇压者通过这些研究获得的信息将是实现利用AbrB基序的独特性质以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.