Regulation of Cytoplasmic RNA Polymerase II

Gene expression is crucial for cell differentiation, development and survival of organisms. It consists of several steps, starting with transcription that is mediated by RNA polymerases. These are protein machineries transcribing and producing different types of RNAs. Although, the individual steps of transcription by RNA polymerase II (Pol II) as well as the structure of Pol II has been extensively studied, surprisingly, there is still little known about its regulation and assembly in cytoplasm. Among the proteins that are important in biogenesis of Pol II are RNA polymerase II associating proteins (RPAP) and small GPN-loop GTPases (GPN). Both of these protein groups were shown to take essential part in assembly of Pol II. The aim of this project was to deepen our knowledge in regulation of Pol II in the cytoplasm as well as the proteins involved in this process. Techniques of structural biology, biochemistry and cell biology were employed to study and characterize cytoplasmic Pol II and its interacting partners. This study shows for the first time the structure of cytoplasmic Pol II at high resolution. The structure also reveals proteins interacting with Pol II in cytoplasm, namely GDOWN1, RPAP2. Comparing the structure of cytoplasmic Pol II with transcribing Pol II revealed striking difference in clamp region that is not in closed state. Furthermore, GDOWN1 and RPAP2 make steric clashes with various transcription factors bound to Pol II during different stages of transcription. Even though GPN1 and GPN3 proteins were not resolved in the cytoplasmic Pol II structure, they are part of the complex and their interaction with Pol II was confirmed in vitro. RPAP2 stabilizes these proteins on Pol II and several experiments suggest that they interact with the clamp region. In addition, GDOWN1, RPAP2 and GPNs might keep clamp in open or partially open state. Based on these results I propose a novel model of regulation of Pol II in cytoplasm. GDOWN1, RPAP2, GPN1 and GPN3 bind to Pol II in cytoplasm and doing so they can prevent pre-mature binding of DNA or RNA and different transcription factors to Pol II in cytoplasm or before engaging in transcription nucleus. This research contributes to the current knowledge of molecular mechanisms of Pol II regulation in cytoplasm.