Determinants and dynamics of RNA polymerase I transcription initiation

蛋白质合成是所有细胞生存和运作的核心。蛋白质生产水平与功能性核糖体--细胞蛋白质工厂--的数量相关，因此生长中的细胞消耗大量能量制造新的核糖体。因此，核糖体生物发生和功能需求的平衡对细胞的健康至关重要。在真核细胞中，核糖体生物发生的限速步骤是由RNA聚合酶I(Pol I)合成核糖体RNA前体(RRNA)；事实上，细胞中60%的RNA是核糖体RNA。POL I受体的活性受控制细胞生长的信号通路的严格调控。这种调节平衡在许多类型的癌症中失去，导致异常的POL I过度活动，从而导致患病细胞中更高的蛋白质产量水平。有趣的是，快速生长的癌细胞对非常有效的Pol I的依赖意味着特定的Pol I抑制剂可能被用来治疗癌症。新的Pol I抑制剂的开发需要对Pol I转录动力学的精确了解，特别是在高度调控的转录起始阶段。由于Pol I的启动涉及具有不同分子构象的多个可逆步骤，因此Pol I转录启动的动力学和决定因素在很大程度上仍然难以捉摸。事实上，经典的生化方法只能提供异质样品的平均行为，而结构方法只能提供静态图像，因此没有动力学。我建议使用单分子生物物理技术，通过观察单个Pol I轨迹来解开多步骤机制的异质性和复杂性。具体地说，我将使用单分子磁钳通过实时监测启动子弯曲和打开的程度和时间来解开Pol I转录启动的动力学。我将进一步将磁镊仪与荧光光谱相结合，同时监测转录起始复合体的组装以及随后启动子构象的变化。我的建议的结果将为了解Pol I转录启动的分子机制提供一个新的角度，并定义启动因子如何调节这一机制，为药物开发开辟新的途径。

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.