Nano-Positioning-System Reveals Structure of the RNA Polymerase II Initially Transcribing Complex (ITC)

Abstract

Transcription initiation is a multi-step process requiring binding of RNA polymerase II (Pol II) to the promoter, melting of DNA and its insertion into the active site. Pol II needs the assistance of several transcription factors (TFs), most essentially the TATA-binding protein (TBP), TFIIB and TFIIF.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. Recently, the structure of a Pol II-TFIIB complex was solved [1] indicating the mechanism of transcription initiation and providing models for closed and open complex. However, the structure of a Pol II initiation complex including all involved TFs is still missing.Here, we employ the recently developed Nano-Positioning-System (NPS) [2] to unravel the structure of a Pol II ITC, consisting of Pol II, TFIIF, TBP, TFIIB and promoter DNA containing a TATA-box, a mismatch bubble and a 7nt RNA transcript. Briefly, by using sm-FRET, we determine distances within double labelled ITCs and the NPS computes 3-dimensional position probability densities of unknown sites on the TATA-box, TBP, TFIIB and the non-template DNA. These results allow us to build a model of Pol II ITC, which is an important intermediate state of Pol II between initiation and elongation. Hence, our model helps understanding the transition mechanism of Pol II from its initiation to its elongation state.[1] Kostrewa et al., Nature 462, 323-330 (2009).[2] Muschielok et al., Nature Methods, 5, 965 (2008).