Determinants and dynamics of RNA polymerase I transcription initiation

• Applicant: David Dulin, Ph.D.
• Subject Area: Biophysics ; Biochemistry
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 448328357

Protein synthesis is at the core of the survival and functioning of all cells. Protein production level correlates with the number of functional ribosomes – the cellular protein factories – and therefore growing cells consume a lot of energy making new ribosomes. Balance in ribosome biogenesis and functional demand is thus essential to cell well-being. In eukaryotic cells, the rate limiting step of ribosome biogenesis is the synthesis of the ribosomal RNA precursor (rRNA) by the RNA polymerase I (Pol I); in fact, 60% of all RNA’s in the cell are ribosomal RNA. The activity of Pol I apparatus is heavily regulated by the signaling pathways that control cell growth. This regulatory balance is lost in many types of cancer, leading to abnormal Pol I hyperactivity, therefore to higher protein production level in sick cells. Interestingly, the dependence of rapidly growing cancer cells on exceedingly efficient Pol I implies that specific Pol I inhibitors might be employed to treat cancer. The development of new Pol I inhibitors requires a precise understanding of Pol I transcription dynamics, in particular during the highly regulated initiation phase of transcription. Because Pol I initiation involves multiple reversible steps with different molecular conformations, the dynamics and the determinants of Pol I transcription initiation have remained largely elusive. Indeed, classical biochemical approaches can only provide the average behavior of a heterogeneous sample, while structural methods offer only static images, hence no kinetics. I propose to use single molecule biophysics techniques that are able to disentangle the heterogeneity and the complexity of multi-steps mechanism by observing individual Pol I trajectories. Specifically, I will use single molecule magnetic tweezers to unravel the dynamics of Pol I transcription initiation by monitoring in real time the extent and timing of promoter bending and opening. I will further combine the magnetic tweezer instrument with fluorescence spectroscopy to simultaneously monitor the assembly of the transcription initiation complex and the consequent changes in the promoter conformation. The outcomes of my proposal will provide a new angle in the fundamental knowledge of the molecular mechanism of Pol I transcription initiation, and define how initiation factors modulate this mechanism, opening new avenues for drug development.

• DFG Programme: Research Grants
• International Connection: Netherlands