Macromolecular Assemblies of the Mammalian Circadian Clock

摘要

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-1 delta (CK1 delta) are present together in an similar to 1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target. Prior to incorporation, CLOCK-BMAL1 exists in an similar to 750-kDa complex. Single-particle electron microscopy (EM) revealed nuclear PER complexes purified from mouse liver to be quasi-spherical similar to 40-nm structures. In the cytoplasm, PERs, CRYs, and CK1 delta were distributed into several complexes of similar to 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 similar to 20-nmand similar to 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.