Structural Studies of RNA Polymerase II Transcription Initiation and Elongation

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PROJECT SUMMARY DNA-directed RNA Polymerase II (Pol II) is one of the most important molecules in biology. Pol II is highly- conserved among eukaryotic organisms and plays a fundamental role in cellular life; specifically, the transcription of genes into messenger RNA. Structural studies of Pol II have been very successful and have allowed snapshots of Pol II in its apo form, in the process of initiation and elongation, during backtracking or paused by DNA lesions. Moreover, single particle cryo-electron microscopy structures of Pol II in complex with the general transcription factors (preinitiation complex), and with elongation factors have provided views of the complexities of the initiation and elongation steps of transcription. A molecular picture of the role that individual factors play during transcription initiation and elongation is beginning to emerge and has generated tremendous progress towards our understanding of gene processing and regulation. Conversely, the molecular details of nucleotide addition and the roles of conformational changes by conserved and essential domains such as the so called “trigger loop” or “bridge helix” in substrate selection and catalysis are not fully understood. Furthermore, pioneering studies are at lower resolution and miss critical information to inform a complete enzymatic mechanism. This project, through extensive technology development and innovation in structural approaches to Pol II, describes the foundation to reveal the active site rearrangements within Pol II leading to catalysis and subsequent translocation. The studies proposed employ a combination of state of the art technologies including time resolved X-ray crystallography, free electron laser and single particle cryo-electron microscopy experiments to elucidate the time evolution of the molecular events during Pol II transcriptional elongation. Importantly, orthogonal approaches are developed to validate results from multiple independent methodologies. The Pol II system represents a model for all cellular RNA polymerases and results obtained will represent foundational models with which Pol I, Pol III, and prokaryotic RNA polymerases may be compared. Insight into the Pol II catalytic mechanism and determination of how activity-altering mutants alter it will provide the framework for understanding ho regulatory factors that target the Pol II active site might work.