Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

中文翻译：

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黄曲霉毒素生物合成调节剂 AflR 和 AflS：DNA 结合亲和力、化学计量和动力学


黄曲霉毒素 (AF) 是由曲霉属真菌产生的强效食源性致癌物，对全世界的健康构成重大风险，并对食物链中的食品安全和生产力提出挑战。迫切需要扰乱 AF 生产、培育抗灾作物和检测受污染食品的新策略。了解 AF 产生的调控机制对于采取有针对性的干预措施以减轻食品和饲料中的毒素积累至关重要。负责 AF 生物合成的基因簇编码生物合成酶和途径特异性调节因子，特别是 AflR 和 AflS。 AflR 是一种 DNA 结合蛋白，可激活簇内的基因转录，而 AflS 通过尚未完全了解的机制增强 AF 的产生。在这项研究中，我们开发了纯化重组 AflR 和 AflS 蛋白的方案，并利用多种测定来表征它们与 DNA 的相互作用。我们的生物物理分析表明 AflR 和 AflS 形成复合物。 AflS 本身没有表现出 DNA 结合能力，但意外地降低了 AflR 的 DNA 结合亲和力。此外，我们发现 AflR 通过一种机制实现其结合特异性，其中两个拷贝的 AflR 或其与 AflS 的复合物以高度协作的方式结合到 DNA 上的靶位点。 AflR、AflS 和 DNA 靶位点相互作用参数的估计值构成了一个基本框架，可以与其他 AF 生物合成调节因子的功能和机制进行比较。