Molecular Structures of Transcribing RNA Polymerase I.

Protein Purification and Complex Assembly Pol I was purified from S. cerevisiae using an AC40 TAP-tag purification protocol as previously described ( Moreno-Morcillo et al., 2014 ), except that purified Pol I was exchanged for EM buffer (150 mM (NH 4 ) 2 SO 4 , 15 mM HEPES-NaOH [pH 7.5], and 10 mM DTT). For the EC1, Pol I was mixed with a 5-fold molar excess of a pre-annealed 38-bp transcription scaffold with an 11-nt mismatch region as previously described ( Hoffmann et al., 2015 ), except that a 20-nt RNA (5′-UAUAUGCAUAAAGACCAGGC-3′) was used. Briefly, the template (T) and non-template (NT) strands were mixed at a final concentration of 50 μM and annealed by heating to 95°C in RNase-free water, and then slowly cooled to 25°C in 1 hr. Then, an equimolar amount of RNA was added and annealed by heating the sample to 45°C, then cooled down to 4°C. The complex was incubated for 1 hr at 4°C to a final concentration of 1 mg/mL in EM buffer. For the OC, EC2, and EC_tWH, Pol I was incubated with a pre-annealed 70-bp transcription scaffold (prepared as described above) containing the core promoter sequence (−50 to +20) (T, 5′-GTCTTCAACTGCTTTCGCATGAAGTACCTCCCAACTACTTTTC CTCACACTTGTACTCCATGACTAAACC-3′; NT, 5′-GGTTTAGTCATGGAGTA CAAGTGTGAGGAAAAGT AGTTGGCGTAGCAGGAGAAGTAAAGCAGTTGAAGAC-3′) and a 15-nt mismatch region with a 10-nt RNA (5′-GAGGUACUUC-3′) in 100 mM potassium acetate, 50 mM HEPES-NaOH (pH 7.5), 5 mM magnesium acetate, and 10 mM DTT. Both mismatch-containing scaffolds are artificial and may differ from an in vivo-created, fully complementary transcription bubble. The sample also contained Pol I-specific transcription factors Rrn3 and core factor, but only a minor fraction of particles contained density corresponding to these proteins.

Sample Preparation

Samples were diluted to 0.2 mg/mL and immediately used for grid preparation. A total of 2.5 μL of sample was applied on freshly glow-discharged Quantifoil grids (400 mesh holey carbon 1.2/1.3 molybdenum for EC1 and 200 mesh holey carbon 2/1 copper for OC, EC2, and EC_tWH) in an FEI Vitrobot Mark II at 20°C and 100% humidity. The sample was incubated for 15 s, blotted for 8 s, and flash frozen in liquid ethane.

Electron Microscopy

Data were acquired on FEI Titan Krios operating at 300 keV through a Gatan Quantum 967 LS energy filter using a 20 eV slit width in zero-loss mode. Movie frames were recorded on a Gatan K2-Summit direct electron detector at a nominal EFTEM (energy-filtered transmission electron microscope) magnification of 105,000× corresponding to 1.35 Å calibrated pixel size (in 4K mode). A total of 715 and 4,235 movie frames were collected for EC1 and OC/EC2/EC_tWH, respectively, using a defocus range of −0.75 to −4 μm. For both datasets, 20 super-resolution frames were collected with a dose rate of 2 e − Å −2 s −1 for a total dose of 40 e − Å −2 . Data collection was fully automated using SerialEM ( Mastronarde, 2005 ).

Image Processing and Model Building

Acquired cryo-EM images were processed using RELION-1.4 ( Scheres, 2012 ), and models were built using COOT ( Emsley and Cowtan, 2004 ) and UCSF Chimera ( Pettersen et al., 2004 ). Figures were prepared using Chimera and PyMol ( Schrodinger, 2010 ). Further details can be found in the Supplemental Experimental Procedures .

RNA Extension Assay The 20-nt and 10-nt

RNA oligonucleotides were radiolabeled with 32 P by T4 PNK and gel purified on denaturing 15% urea-PAGE, for reactions using either the 38-bp or the 70-bp transcription scaffold. A total of 2 pmol of pre-annealed transcription scaffold was incubated with 4 pmol Pol I for 20 min at 20°C in EM buffer in the presence of 5 mM MgCl 2 , and the reaction was initiated by adding the corresponding NTP(s) at a final concentration of 250 μM. RNA extension was performed at 28°C for 20 min. The reaction was stopped by adding loading buffer (8 M urea, TBE) and heating for 2 min at 95°C. The resulting RNA product was analyzed on a denaturing polyacrylamide gel (17% PAGE, 8 M urea) using an FLA7000 phosphoimager (Fujifilm).

Supplemental Information Document S1. Supplemental Experimental Procedures and Figures S1–S4 Document S2. Article plus Supplemental Information