Dynamic Architecture of the RNA Polymerase II Open Promoter Complex

Abstract

Transcription initiation of eukaryotic protein-coding genes is a multi-step process that requires assembly of RNA polymerase (Pol) II and the general transcription factors into the closed complex. Then, the DNA surrounding the TSS is melted and inserted into the Pol II active center cleft to form the open complex (OC), the starting point for RNA synthesis. The OC is hence a central intermediate in the initiation-elongation transition and insights into its structure are crucial for understanding the molecular mechanism of transcription initiation.X-ray crystallography has provided detailed insights into the architecture of Pol II in different functional states, but structural studies of transcription initiation have been hindered by the flexibility of Pol II initiation complexes. As a consequence, the structure of the OC remains unknown.Here, we employ single-molecule Forster Resonance Energy Transfer (smFRET) and Nano-Positioning System (NPS)analysis [1] to determine the 3-dimensional architecture of a minimal OC consisting of Pol II, promoter DNA, TBP, TFIIB, and -IIF. Briefly, by using smFRET, we measure distances within double labeled OCs and the NPS computes 3-dimensional position probability densities of unknown sites on upstream DNA, TATA-DNA, TBP, and TFIIB, which allows us to build a model of the OC.We show that in the OC, TBP and TATA-DNA reside above the Pol II cleft between clamp and protrusion. Downstream DNA is dynamically loaded into the cleft and unloaded from the cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it was located in the closed complex. Hence, our results define the overall structural changes during the initiation-elongation transition, and reveal a key role for the intrinsic flexibility of TFIIB in accommodating these changes.[1] Muschielok et al., Nature Methods, 5, 965 (2008).