BACKGROUND Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. RESULTS Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. CONCLUSION The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins.

中文翻译：

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生物钟蛋白 PER2 和 CRY 与 BMAL1 和 CLOCK 的相互作用。

背景时钟控制基因表达的昼夜节律振荡主要在转录水平上调节。CLOCK 和 BMAL1 的异二聚体作为靶基因转录的激活剂；然而，PER 和 CRY 蛋白与异源二聚体的相互作用消除了其转录激活能力。因此，PER 和 CRY 被称为生物钟的负调节器。为了进一步阐明时钟的正负成分如何相互作用的机制，我们使用哺乳动物双杂交系统和共免疫沉淀分析表征了 PER2、CRY1 和 CRY2 与 BMAL1 和 CLOCK 的相互作用。结果 发现 PER2 和 CRY 蛋白都与 BMAL1 相互作用，而只有 PER2 与 CLOCK 相互作用。CRY 蛋白似乎比 PER2 对 BMAL1 具有更高的亲和力。而且，我们提供证据表明 PER2、CRY1 和 CRY2 与 BMAL1 蛋白中的不同结构域结合。结论 时钟控制转录 PER2、CRY1 和 CRY2 的调节因子与 BMAL1-CLOCK 异源二聚体的每个单一成分相互作用的能力不同，在 BMAL1 的情况下，它们的相互作用位点也不同。我们的数据支持以下假设：CRY 蛋白，尤其是 CRY1，是比 PER 蛋白更强的阻遏物。