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.
Highlights
The eukaryotic genome is transcribed by three RNA polymerases
Straight and bent bridge helix (BH) conformations have been observed in bacterial Pol (Tuske et al, 2005), and limited movements of the BH during translocation have been observed in Pol II (Brueckner and Cramer, 2008; Wang et al, 2006), consistent with complementary mutagenesis and molecular dynamics studies in different RNA polymerases
DNA-Binding Cleft of polymerase I (Pol I) Closes during Elongation Complex Formation To investigate the structural changes that Pol I undergoes upon initiation and elongation, we assembled two Pol I complexes with different transcription scaffolds: a 38-bp transcription scaffold containing an 11-nt transcription bubble and a 20-nt RNA oligonucleotide, as previously described (Hoffmann et al, 2015), and a longer 70-bp transcription scaffold containing the wild-type rDNA promoter sequence with a 15-nt transcription bubble and a 10-nt RNA oligonucleotide (Experimental Procedures)
Summary
Hoffmann, ..., Wim J.H. Hagen, Carsten Sachse, Christoph W. Tafur et al present cryo-EM structures of transcribing Pol I that reveal the conformational changes required for rendering the enzyme active. The DNA-binding cleft narrows, the bridge helix folds, and the A12.2 C-terminal domain is excluded from the active site. 5M5W 5M5X 5M5Y 5M64 d During elongation, the Pol I A12.2 C-terminal domain is excluded from the cleft d A49 tandem winged helix domain contacts upstream DNA, similar to TFIIE. 2016, Molecular Cell 64, 1135–1143 December 15, 2016 a 2016 The Author(s).
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