Molecular Structures of Transcribing RNA Polymerase I

Abstract

SummaryRNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.

Structural insights into transcription initiation by yeast RNA polymerase I

“…These findings also agree with previous structures showing that Pol I and Pol II can position downstream DNA correctly in the absence of transcription factors and RNA (Cheung et al , 2011; Tafur et al , 2016). …”

“…Most prominently, upstream DNA is positioned differently through interactions with the Rrn7 cyclin domains and the DBHB, which appear to maintain the upstream DNA next to the Pol I protrusion and wall in a position close to that observed for the EC (Tafur et al , 2016). This could represent a functional adaptation of Pol I toward a more efficient transcription initiation mechanism where the transition from initiation to elongation is facilitated, thereby increasing the efficiency of promoter escape (Schneider, 2012).…”

“…We stepwise assembled the minimal Pol I PIC by adding recombinant CF, Rrn3, and natively purified, transcriptionally active Pol I to a 70 base pair (bp) transcription scaffold (−50 to +20) containing the core rDNA promoter (−38 to +5) with a 15‐nucleotide (nt) mismatch and a 10‐nt RNA (Tafur et al , 2016). A homogenous sample containing the 18‐subunit protein complex with a molecular mass of ~900 kDa was obtained using dialysis from high to low salt, whereas the complex did not form in the absence of DNA (Appendix Fig S1A–D, see Materials and Methods).…”

“…We collected 4,235 movie frames on a FEI Titan Krios equipped with a Gatan K2 Summit direct electron detector and processed them using RELION (Scheres, 2012). Initial analysis revealed that most of the complex dissociated and the majority of the particles corresponded to Pol I bound to DNA (Tafur et al , 2016). Extensive particle classification allowed excluding particles containing only Pol I or Pol I‐Rrn3 and improved the density corresponding to CF (Appendix Fig S2), leading to 38,589 Pol I initiation complex particles that were refined to an average resolution of 4.4 Å (Figs 1 and EV1A).…”

“…Subsequently, 1.5 μM of CF was added in a buffer with 300 mM potassium acetate, 50 mM HEPES‐NaOH pH 7.5, 5 mM magnesium acetate, and 10 mM DTT and dialyzed overnight against 100 mM potassium acetate, 50 mM HEPES‐NaOH pH 7.5, 5 mM magnesium acetate, and 10 mM DTT. Pol I was tested to be active in an RNA extension assay with this template (Tafur et al , 2016). …”