Abstract
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
Highlights
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn[3] on the rDNA core promoter for transcription initiation
We present previously uncharacterized structures of Pol I closed complex (CC), Pol I open complex (OC) intermediates and Pol I OCs combined with biochemical data that further rationalize promoter recognition by CF and Pol I
Our structures illustrate the major structural rearrangements that result in spontaneous DNA opening and OC formation during Pol I transcription initiation (Supplementary Movie 1), thereby further completing the ‘ratcheting’ model proposed for Pol I transcription initiation[10]
Summary
RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn[3] on the rDNA core promoter for transcription initiation. We report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. The limited resolution of the Pol I IC structures[9,10,11] and lack of complementary biochemical data precluded a more detailed understanding of how CF subunits recognize promoter DNA and facilitate DNA opening in an ATP-independent manner. The different reconstructions provide mechanistic insights into complex assembly, allow following template and non-template DNA strand paths, show the necessary conformational changes during promoter opening and reveal the interplay between Rrn[3] and A49 tWH in promoter escape. The structural data, combined with biochemical experiments, allow better understanding the specificity of promoter recognition by CF and A49 tWH
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