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Dynamic architecture of the RNA polymerase I transcription initiation machinery

Final Report Summary - POL1PIC (Dynamic architecture of the RNA polymerase I transcription initiation machinery)

RNA polymerase (Pol) I and III synthesize non-translated RNAs critically required for ribosome assembly and protein synthesis. While Pol I is specialized in pre-ribosomal RNA synthesis as a first step in ribosome biogenesis, Pol III synthesizes 5S ribosomal RNA in addition to tRNAs and other small, structured RNAs. Many protein kinases, transcription factors and tumor suppressors directly target the Pol I and Pol III transcription machineries carefully regulating their activities in healthy cells, while mis-regulation of Pol I and Pol III transcription is observed in a variety of cancers. During the project, we studied the structure and dynamics of yeast Pol I and its pre-initiation complex using cryo-electron microscopy as primary structural biology technique combined with chemical cross-linking/mass spectrometry, in vitro biochemical transcription assays and single-molecule fluorescence light microscopy.

At the start of the project, crystal structures of unbound 14-subunit yeast Pol I had just been solved by us and the Cramer group. This milestone achievement formed the starting point to determine cryo-EM structures of elongating Pol I bound to a transcription scaffold. Comparison with unbound Pol I showed gradual closing of the DNA-binding cleft and folding of the “bridge helix” during DNA binding. Subsequently, we (and others) determined the cryo-EM structure of a minimal yeast Pol I pre-initiation complex comprising Pol I, three-subunit core factor and bridging factor Rrn3 bound to minimal Pol I DNA promoter that showed an unexpected architecture with TFIIB-like factor Rrn7 binding at an upstream position to DNA compared to the TFIIB-binding site in the Pol II PIC. The initial Pol I PIC structure was subsequently followed up by several higher resolution cryo-EM structures of Pol I PICs at different states that showed the dynamic transition from a closed to an open transcription complex during promoter melting and transcription bubble formation. Current efforts are focusing now on reconstituting a complete Pol I PIC that in addition to core factor and Rrn3 also comprises upstream-activating factor (UAF) and TBP. In a parallel effort, we also obtained the cryo-EM structure of 17-subunit Pol III unbound and bound to a transcription scaffold at near-atomic resolution. This major achievement allowed us to complete the gallery of eukaryotic, multi-subunit DNA-dependent RNA polymerases more than 10 years after the first X-ray structure determination of Pol II and the Pol II elongation complex. The Pol III cryo-EM structure also laid the basis for the cryo-EM structure determination of a minimal Pol III PIC that comprises Pol III, TFIIIB with TBP, TFIIB-related factor Brf1 and Bdp1. We obtained cryo-EM structures in closed and open conformations that allowed suggesting a mechanism for promoter melting by Pol III and to visualize this process in a ‘movie’.

Thanks to support by the POL1PIC ERC Advanced Grant, we have obtained the molecular structures of Pol I and Pol III in different functional states, but also their minimal preinitiation complexes. As a result, the broad communities interested in structural biology and transcriptional regulation can now compare similarities and differences between the Pol I, Pol II and Pol III transcription machineries thereby gaining detailed insight into their molecular mechanisms, but also the evolutionary relationships between the eukaryotic RNA polymerases I, II and III but also archaea and bacterial RNA polymerases. Beyond given insight into their basic mechanisms and architecture, the first molecular structures of Pol and Pol III and their PICs also provide excellent starting points for now studying their interactions with cellular regulators, but also control their function with small molecule effectors.