Widespread epistasis shapes RNA Polymerase active sites function and evolution

Multi-subunit RNA Polymerases are responsible for transcription in all kingdoms of life. At the heart of these msRNAPs is an ultra-conserved active site domain, the trigger loop, coordinating transcription speed and fidelity by critical conformational changes impacting multiple steps in substrate selection, catalysis and translocation. Previous studies have observed several different types of genetic interactions between eukaryotic RNA polymerase II TL residues, suggesting that the TL function is shaped by functional interactions of residues within and around the TL. The extent of these interaction networks and how they control msRNAP function and evolution remain to be determined. We aim to comprehensively dissect the TL interaction landscapes by deep mutational scanning in three eukaryotic msRNAPs of Saccharomyces cerevisiae. Our studies provide a powerful system to understand the plasticity of RNA polymerase mechanism and evolution.多亚基RNA聚合酶（Multi-subunit RNA Polymerases）负责所有生物界的转录过程。这类msRNAP的核心为一个高度保守的活性位点结构域——触发环（trigger loop），其通过影响底物选择、催化及移位等多个步骤的关键构象变化，协调转录的速度与保真度。既往研究已在真核RNA聚合酶II的TL残基间观测到多种不同类型的遗传相互作用，提示TL的功能由TL内部及周边残基的功能相互作用所塑造。然而，这类相互作用网络的覆盖范围，以及它们如何调控msRNAP的功能与进化，仍有待阐明。本研究旨在通过对酿酒酵母（Saccharomyces cerevisiae）的三种真核msRNAP开展深度突变扫描（deep mutational scanning），全面解析TL的相互作用图谱。本研究将为理解RNA聚合酶作用机制的可塑性及其进化过程提供强有力的研究体系。