Final Activity Report Summary - RNA POLYMERASE III (Structural studies of yeast RNA polymerase III)
During gene transcription, RNA is synthesised from a DNA template by DNA-dependent RNA polymerases (Pols). Eukaryotic cells contain three nuclear RNA polymerases, which are responsible for the synthesis of ribosomal RNA (Pol I), pre-messenger RNA (Pol II) and small RNAs including transfer RNAs (Pol III). The three RNA polymerases differ in subunit composition, reflecting their evolutionary specialisation in transcription of different genes and suggesting their independent regulation. Genes of different classes differ in promoter structure, which dictates assembly of the specific Pol with class-specific general transcription factors (initiation factors) into a preinitiation complex. With a total molecular weight of nearly 700 kDa, Pol III is the largest and the most complex nuclear RNA polymerase.
As stated in the original working plan, we managed to establish large-scale fermentation (200 liters) and purification of Pol III from S. cerevisiae. By means of cryo-electron microscopy and crystallography (hybrid approach) we were able to obtain three independent snapshot of Pol III: first, the apo-enzyme in isolation, second, the complex with a specific Pol III gene (elongation complex) and third, a complex with the global negative regulator Maf1. These reconstructions are of high quality and allowed us to rationalize the specific features of the Pol III enzyme and its activity, supporting previously published biochemical data, now on a structural level.
Upon completion of functional in vitro data experiments to corroborate our model, we will be able to publish our findings in a high-impact journal, given the importance of such basic mechanisms that are of central importance for the whole transcription regulation community.
As stated in the original working plan, we managed to establish large-scale fermentation (200 liters) and purification of Pol III from S. cerevisiae. By means of cryo-electron microscopy and crystallography (hybrid approach) we were able to obtain three independent snapshot of Pol III: first, the apo-enzyme in isolation, second, the complex with a specific Pol III gene (elongation complex) and third, a complex with the global negative regulator Maf1. These reconstructions are of high quality and allowed us to rationalize the specific features of the Pol III enzyme and its activity, supporting previously published biochemical data, now on a structural level.
Upon completion of functional in vitro data experiments to corroborate our model, we will be able to publish our findings in a high-impact journal, given the importance of such basic mechanisms that are of central importance for the whole transcription regulation community.