The Conserved Strategies of RNA Polymerase I Activation and Hibernation in Yeast

Within this work, the question whether the mechanisms of deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) Polymerase I (Pol I) activation and hibernation are conserved in yeast has been addressed. Pol I is a specialised enzyme that transcribes ribosomal RNA (rRNA) genes in all eukaryotes. Until recently, all published eukaryotic Pol I structures have been solved from the baker’s yeast Saccharomyces cerevisiae (S. cerevisiae). Thus far, the storage of Pol I in an inactive dimeric conformation relies on S. cerevisiae-specific elements such as the ‘connector’. Furthermore, it is unclear whether structural elements such as the ‘expander’ operate similarly in homologous enzymes from other organisms. Enzymatic activation by ‘cleft’ contraction following the rotation of the central polymerase modules is believed to be of paramount importance but was not yet observed in any other species. Like S. cerevisiae, the fission yeast Schizosaccharomyces pombe (S. pombe) is an equally valuable model organism bridging the evolutionary gap towards Homo sapiens (H. sapiens) and was thus chosen for this investigation. To answer the research questions, Pol I from S. pombe was extracted and purified for subsequent structural and functional characterisation by single-particle electron cryo-microscopy (cryo-EM) and in vitro transcription assays. This work provides the first published structures of Pol I from an organism other than S. cerevisiae in an inactive monomeric and an actively elongating state solved at high resolution using single-particle cryo-EM. Surprisingly, it was found that Pol I dimerization can occur independently of the A43 ‘connector’ domain and the cryo-EM reconstruction of such a dimer could be solved. The architecture of S. cerevisiae and S. pombe Pol I is similar, despite poor overall sequence identity. Comparative analysis of structural models derived from high-resolution reconstructions shows that a conserved contraction of the active centre cleft accomplishes activation. Overall, the results of this work support the hypotheses of Pol I activation by contraction and hibernation by dimerization. Only recently, new insights into the H. sapiens Pol I have been made available, allowing for an even more thorough discussion of the evolutionary conservation of structural features.