AbrB binding to developmentally controlled promoters

描述(申请人提供)：枯草芽孢杆菌ABRB蛋白是一种关键的全球调节因子，它调节基因的表达，以适应在次优环境、面对压力和孢子形成发育过程的初始阶段的代谢需要。除了在快速生长过程中阻止稳定期相关功能的不适当表达外，有证据表明，AbrB还在调节分解代谢抑制方面发挥作用，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末端结构域对一类新的DNA结合基序具有聚集性，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。超过40个操纵子编码广泛的代谢功能，包括必需的产孢子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、梭状芽孢杆菌、李斯特菌和嗜酸杆菌属物种(包括致病物种)中，至少还有25种调节蛋白与AbrB的DNA结合域具有广泛的氨基酸同源性。阐明导致灵活的AbrB结合特异性的因素将有助于深入了解蛋白质-DNA识别机制，以及细胞如何经济地利用单一蛋白质来协调各种应激反应和发育选择。研究蛋白质中特定残基和区域所起的作用，比较和对比AbrB同源物中序列变化所指定的性质，将为具有这种灵活结合基序的蛋白质利用大分子相互作用实现广泛而特异的DNA识别特性提供重要的见解。AbrB结构与DNA结合特性的关系，以及存在于结合表面及其附近的特定氨基酸残基所起的确切作用，将通过突变选择、突变分析和对选定的AbrB同源物的结合结构域的检查来探索。这些蛋白质的羧基末端结构域主要是高阶多聚化结构域的假设将通过各种方法进行验证，包括对突变蛋白的遗传、生化和生物物理分析，以及构建具有完整或截断的C结构域与其他AbrB同源物的DNA结合结构域或与？抑制者。通过这些研究获得的信息将是迈向最终目标的关键一步，即利用AbrB基序的独特性质来设计或直接选择与任何所需DNA靶标特异结合的特定变体。

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.