Structure of human cytoplasmic Pol II complex explains global transcription repression by GDOWN1
The assembly and nuclear import of RNA polymerase II (Pol II) are not fully understood. Here, the authors identify GDOWN1’s role in Pol II assembly and transcription regulation using biochemical and cryo-EM analyses of c
RNA polymerase II (Pol II) is a 12-subunit enzyme crucial for gene transcription in the nucleus. However, its assembly in the cytoplasm, nuclear import, and nuclear function of assembly factors remain poorly understood. Here, we isolated Pol II from the cytoplasmic fraction of human cells (cfPol II) and found it associated with the assembly and transport factors GDOWN1, RPAP2, GPN1, and GPN3. Cryo-EM analysis of cfPol II resolved RPAP2 and GDOWN1 bound to Pol II at 2.9 Å resolution and showed that Pol II was fully assembled in the cytoplasm before nuclear import. The structure of GDOWN1 bound to the Pol II surface revealed three distinct regions of GDOWN1 that interacted with the RPB2 protrusion domain, RPB3, and RPB10. Biochemical analyses showed that GDOWN1 facilitated the soluble expression of a subcomplex comprising RPB3, RPB10, RPB11, and RPB12, suggesting a role for GDOWN1 in Pol II assembly. Furthermore, GDOWN1 binding to Pol II overlapped with binding sites of the essential transcription factors IIB and IIF, rendering cfPol II inactive in promoter-dependent transcription initiation in vitro. The authors' results provided a basis for GDOWN1-dependent global transcription repression and suggested a model for a role of GDOWN1 in Pol II assembly, import, and transcription regulation.