O'Neill E, Ng L C, Sze C C, Shingler V
Department of Cell and Molecular Biology, Umeå University, Sweden.
Mol Microbiol. 1998 Apr;28(1):131-41. doi: 10.1046/j.1365-2958.1998.00780.x.
The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.
源自铜绿假单胞菌的σ54依赖性调节因子DmpR具有一个氨基末端A结构域，该结构域控制芳香效应物激活的特异性；一个中央C结构域，介导转录激活所必需的ATP酶活性；以及一个参与DNA结合的羧基末端D结构域。在芳香效应物存在的情况下，DmpR蛋白促进来自-24、-12 Po启动子的转录，该启动子控制着特殊（甲基）苯酚分解代谢酶的表达。先前对DmpR的分析得出了一个模型，其中A结构域作为DmpR的ATP酶和转录促进特性的结构域间阻遏物，直到结合特定的芳香效应物。在这里，通过将DmpR的部分片段表达为独立的多肽，剖析了A结构域发挥其生物学功能的自主性。结果表明，DmpR的A结构域对于结合苯酚既是必需的也是充分的。对苯酚结合的分析表明，DmpR每个单体有一个结合位点，解离常数为16微摩尔。还表明A结构域对C结构域具有特异性亲和力，并在体外自主抑制C结构域介导的ATP酶活性。然而，A结构域与调节因子其余部分的物理解偶联导致了一个系统，该系统不能通过其正常的去阻遏机制对芳香族化合物作出反应。本文讨论了自主表达的A结构域尽管具有结合苯酚的能力，但对芳香族化合物无反应的机制意义。
