Final Report Summary - TF3B_P3 (Structural characterization of TFIIIB and its involvement in RNA Pol III transcription) 1. FINAL PUBLISHABLE SUMMARY REPORT Transcription is a highly regulated process that allows synthesising RNA from DNA. This process is crucial for cells to adapt during different stages of development and to changes in their environmental conditions. In eukaryotes three different molecular machines participate in the transcription of specific classes of genes. RNA Polymerase (Pol) II transcribes the largest number of target genes including all protein-coding genes. Pol III is the largest of the RNA polymerases and synthesises the transfer RNA that carries the protein building blocks to the ribosome for assembly. Pol I transcribes one gene encoding the precursor ribosomal RNA that is later processed to become an essential component of ribosomes. Since the ribosome represent between 15% and 20% of the cell weight, the activity of Pol I can reach 60% of cellular RNA synthesis to produce approximately 2,000 ribosomes per minute. Scientists have known for over a decade what RNA polymerase II looks like and how it works, but obtaining detailed information on the structures of its counterparts has proven extremely difficult. Besides, in recent years, the tight regulation of Pol I and Pol III during cell growth and division has been demonstrated. Therefore, defects in Pol I and Pol III regulation are associated with alterations in cellular proliferation and, thus, with tumour development.Our group has deciphered part of the transcription puzzle by determining the three-dimensional structure of Pol I. By solving the structure of this machine we have found that it has permanently incorporated additional subunits as functional modules that are transiently recruited in other Pols. As there is no need to recruit external factors, things are simpler. On one side, the fact that RNA polymerase I brings those helper modules permanently on board could explain why this enzyme is faster than their counterparts. On the other side, the cell has fewer ways of controlling RNA polymerase I’s activity, since it can’t influence it by changing the availability of helper proteins. But here, RNA polymerase I’s regulation provides a solution. The structure showed that this molecular machine has a built-in regulatory mechanism: it can stop itself by bending a loop in its DNA binding site to block the space the DNA would occupy. In general, the findings, published in Nature (Fenández-Tornero C.*, Moreno-Morcillo M.*, et al. Nature, 2013 (*equal contribution)), describe the specific characteristics observed in the structure of Pol I that point at the specific role of this enzyme and opens the door to finding new antitumor drugs.In order to obtain the final model of Pol I, the enzyme needed to be isolated and crystallized. The crystallization of such big complex was a very challenging process. Given the large size and dynamic of this enzyme, advanced methods for crystallization and structural determination through X-ray crystallography were required. The unconventional approaches employed to solve the structure were also published in a second manuscript (Moreno-Morcillo M. et al., Acta Crystallographica Section D, 2014), where we reported the crucial steps that were undertaken to successfully build the atomic model of this multi-subunit enzyme.The work was carried out in collaboration with Carlos Fernández-Tornero’s lab at the Centro de Investigaciones Biológicas in Madrid, Spain, as well as with researchers at the University of Gӧttingen (Germany) and the SOLEIL synchrotron (France). Most of the structural data were collected at the just mentioned synchrotron and at the Petra III ring at EMBL Hamburg (Germany).Furthermore, combination of “hybrid approaches” (cryo-EM and chemical cross-linking coupled to MS) has allowed getting insights into the three-dimensional model of Pol III. This information should allow us to understand the overall architecture and dynamics of this assembly and will inform us about interaction interfaces that could to be targeted for future studies on drug design. Two publications related to the 3D model of Pol III are in preparation. All together our results corroborate the emerging view that the basic architecture of Pol I, Pol II and Pol III and their specific pre-initiation complexes is conserved. As an even more interesting aspect, we demonstrated that small differences among the three polymerases are sufficient to determine their specificities. Therefore, our work stimulates future research by providing clues that can support the design of specific Pol I, II or III inhibitors with therapeutic potential.