The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an ∼750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical ∼40-nm structures. In the cytoplasm, PERs, CRYs, and CK1δ were distributed into several complexes of ∼0.9–1.1 MDa that appear to constitute an assembly pathway regulated by GAPVD1, a cytoplasmic trafficking factor. Single-particle EM of two purified cytoplasmic PER complexes revealed ∼20-nm and ∼25-nm structures, respectively, characterized by flexibly tethered globular domains. Our results define the macromolecular assemblies comprising the circadian feedback loop and provide an initial structural view of endogenous eukaryotic clock machinery.

哺乳动物的生物钟建立在反馈环的基础上，其中 PER 和 CRY 蛋白抑制它们自身的转录。我们发现，在小鼠肝细胞核中，所有三种 PER、CRY 和酪蛋白激酶-1δ (CK1δ) 一起存在于~1.9-MDa 阻遏物组件中，该组件定量地整合了其 CLOCK-BMAL1 转录因子靶标。在掺入之前，CLOCK-BMAL1 存在于约 750 kDa 的复合物中。单粒子电子显微镜 (EM) 显示从小鼠肝脏纯化的核 PER 复合物为准球形~40 nm 结构。在细胞质中，PER、CRY 和 CK1δ 分布在~0.9-1.1 MDa 的多个复合物中，这些复合物似乎构成了由细胞质运输因子 GAPVD1 调节的组装途径。两种纯化的细胞质 PER 复合物的单粒子 EM 分别显示了~20 nm 和~25 nm 的结构，其特征是灵活束缚的球状结构域。我们的结果定义了包含昼夜节律反馈环的大分子组装体，并提供了内源性真核生物钟机制的初始结构视图。