Structure of cytoplasmic RNA polymerase II Hlavata, Annamaria Neuditschko, Benjamin Schellhaas, Ulla Plaschka, Clemens Herzog, Franz Bernecky, Carrie A ; https://orcid.org/0000-0003-0893-7036 RNA polymerase II (Pol II) must be assembled in the cytoplasm before it enters the nucleus, where it transcribes protein-coding genes. Although transcription by Pol II is intensively studied, how this central multi-subunit enzyme is made and the role of dedicated factors remains unclear. Here, we report the integrative structural analysis of a native human Pol II from the cytoplasm captured near the end of biogenesis. The complex contained Gdown1 and three biogenesis factors – RPAP2 and the critical small GTPases GPN1 and GPN3. Cryo-EM analysis of the complex revealed how Gdown1 and RPAP2 associate with Pol II and prevent the premature association of transcription factors. Further biochemical and cryo-EM analysis revealed how RPAP2 recruits GPN1–GPN3 to the complex, and how the assembly of the RPAP2–GPN1–GPN3 complex is controlled by GTP hydrolysis. The combined results uncover a network of interactions that chaperone cytoplasmic Pol II to prevent aberrant interactions, reveal a GTP-controlled switch during the final stages of Pol II biogenesis, and suggest a general mechanism for the action of GPN-loop GTPase family of enzymes. bioRxiv 2025 info:eu-repo/semantics/preprint doc-type:preprint text http://purl.org/coar/resource_type/c_816b https://research-explorer.ista.ac.at/record/20804 Hlavata A, Neuditschko B, Schellhaas U, Plaschka C, Herzog F, Bernecky C. Structure of cytoplasmic RNA polymerase II. 2025. doi:<a href="https://doi.org/10.64898/2025.12.10.692585">10.64898/2025.12.10.692585</a> eng info:eu-repo/semantics/altIdentifier/doi/10.64898/2025.12.10.692585 info:eu-repo/semantics/openAccess