Working on this project:
Over the 2nd funding period our first contribution was the long-sought structure of the active, transcribing RNA Polymerase I (Pol I) enzyme. With single-particle cryo-electron microscopy (cryo-EM), we solved the structure at atomic resolution and by cryo-electron tomography (cryo-ET) we visualized the transcription machinery with the actively transcribing Pol I on rRNA genes. For the latter, we used a special preparation technique (“Miller-type spreads”), which allowed to spread the nucleoplasm into a thin layer that made it accessible for electron microscopy techniques. Our second contribution were substantial advances in image processing, which improved the image quality such that we can now address the molecular sociology of the transcription in small cells like bacteria. Our third contribution were detailed mutational studies in mycoplasma that now allow us to pursue our ultimate goal, the deciphering of the transcription machinery in vivo. Altogether in the 3rd funding period, we aim to analyze the conformational states of RNA polymerases and understand the regulation of pausing, backtracking and in particular termination under native conditions in our bacterial and eukaryotic systems. Latest discoveries show that the bacterial and eukaryotic RNA polymerases adopt a comparable active center conformation during transcription elongation. The next frontier is to understand the regulation of the enzymes in the other states. Thus, for the third funding period we aim to visualize the enzymes on the genes in order to understand the regulation of pausing, backtracking and in particular termination under native conditions in our bacterial and eukaryotic systems. Using ‘Miller-type’ spreads of the RNA Polymerase III (Pol III) transcribing 5S rRNA genes. We will visualize arrays of Pol III containing initiation, elongation and termination states. The resolutions of the cryo-electron tomograms will enable the analysis of the conformational states and interaction partners while attached to the DNA and the nascent rRNA. The importance of this study is two-fold: (a) One could create an unprecedented all-states movie of a complete RNA Polymerase transcription cycle, which given the evolutionary conservation of the RNA polymerases will allow important insights to all other RNA polymerases and (b) the data could provide the structural basis for the studies connecting malregulation of Pol III to tumorigenesis. In parallel, we will also visualize the bacterial RNA polymerase (RNAP) in vivo within mycoplasma cells also by ‘Miller-type’ spreads. These bacteria pose due to their tiny size favorable imaging properties for electron tomography and importantly they have one of the smallest self-replicating genomes. This simplicity holds promise to decipher the absolute basic mechanisms of the transcription/translation apparatus in whole cells. In particular, we would like to: (a) see and quantify cellular polymerases and their molecular sociology including ribosomes, (b) analyze differences in pausing/termination of the RNAP between non-coding versus protein-coding genes deliberately engineered to give raise to known identical nascent RNA secondary structures (hairpins). Since in bacteria transcription on protein-coding genes is tightly coupled to expressome/polysome formation, our aim is to depict variations and common RNA regulation themes in pausing and termination of the RNAP which is not possible in approaches using highly purified components given the sensitivity of the system to perturbations of the molecular sociology (Cabrera and Jin 2003).