Abstract
Recent X-ray crystallographic studies of Pol II in complex with the general transcription factor (GTF) IIB have begun to provide insights into the mechanism of transcription initiation. These structures have also shed light on the architecture of the transcription preinitiation complex (PIC). However, structural characterization of a functional PIC is still lacking, and even the topological arrangement of the GTFs in the Pol II complex is a matter of contention. We have extended our activity-based affinity crosslinking studies, initially developed to investigate the interaction of bacterial RNA polymerase with σ, to the eukaryotic transcription machinery. Towards that end, we sought to identify GTFs that are within the Pol II active site in a functioning PIC. We provide biochemical evidence that TFIIB is located within ∼9 Å of the −2 site of promoter DNA, where it is positioned to play a role in de novo transcription initiation.
Highlights
Multisubunit RNA polymerases (RNAPs) display a conserved core structure across the three domains of life
Our general strategy was to use super-selective crosslinking and labeling techniques to define the functional topography of the Pol II active center, similar to what has previously been described for prokaryotic systems [11, 49]
There is not a general agreement on the dispensability of TFIIH for abortive initiation since it may be dependent on specific experimental conditions, as others have reported a requirement for TFIIH in abortive initiation [55]
Summary
Multisubunit RNA polymerases (RNAPs) display a conserved core structure across the three domains of life. While one RNAP suffices for all RNA synthesis in eubacteria and archaebacteria, eukaryotic cells harbor three distinct enzymes, RNAPs I, II, and III (Pols I, II, and III). Counterparts of all five of the core bacterial RNAP subunits are found in the eukaryotic Pols I, II, and III [2]. These orthologs include the two largest subunits RPB1 and RPB2, which correspond, respectively, to the bacterial β' and β subunits [2,3,4], RPB3 and RPB11, which correspond to the two copies of the bacterial α subunit [5], as well as the small RPB6 subunit, which corresponds to the bacterial ω subunit [6]. Consistent with the increased complexity of the eukaryotic transcriptional machinery, Pol II has several additional subunits that do not have bacterial counterparts
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