preprint published Annamaria Hlavata author 36062FEC-F248-11E8-B48F-1D18A9856A87 Benjamin Neuditschko author Ulla Schellhaas author Clemens Plaschka author Franz Herzog author Carrie A Bernecky author 2CB9DFE2-F248-11E8-B48F-1D18A9856A870000-0003-0893-7036 CaBe department 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. bioRxiv2025 eng 10.64898/2025.12.10.692585 A. Hlavata, B. Neuditschko, U. Schellhaas, C. Plaschka, F. Herzog, and C. Bernecky, “Structure of cytoplasmic RNA polymerase II.” bioRxiv, 2025. Hlavata, Annamaria, Benjamin Neuditschko, Ulla Schellhaas, Clemens Plaschka, Franz Herzog, and Carrie Bernecky. “Structure of Cytoplasmic RNA Polymerase II.” bioRxiv, 2025. <a href="https://doi.org/10.64898/2025.12.10.692585">https://doi.org/10.64898/2025.12.10.692585</a>. Hlavata, A., Neuditschko, B., Schellhaas, U., Plaschka, C., Herzog, F., &#38; Bernecky, C. (2025). Structure of cytoplasmic RNA polymerase II. bioRxiv. <a href="https://doi.org/10.64898/2025.12.10.692585">https://doi.org/10.64898/2025.12.10.692585</a> Hlavata A, Neuditschko B, Schellhaas U, Plaschka C, Herzog F, Bernecky C. 2025. Structure of cytoplasmic RNA polymerase II. <a href="https://doi.org/10.64898/2025.12.10.692585">10.64898/2025.12.10.692585</a>. Hlavata, Annamaria, et al. <i>Structure of Cytoplasmic RNA Polymerase II</i>. bioRxiv, 2025, doi:<a href="https://doi.org/10.64898/2025.12.10.692585">10.64898/2025.12.10.692585</a>. 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> A. Hlavata, B. Neuditschko, U. Schellhaas, C. Plaschka, F. Herzog, C. Bernecky, (2025). 208042025-12-11T13:33:27Z2025-12-15T09:48:22Z