Abstract
Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82–C34–C31 heterotrimer in close proximity to the stalk. The C53–C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.
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The 3.9 Å cryo-EM map of elongating Pol III and the two maps of apo Pol III have been deposited in the Electron Microscopy Data Bank with accession codes EMD-3178 (elongating Pol III) and EMD-3179 (‘closed clamp’ apo Pol III) EMD-3180 (‘open clamp’ apo Pol III). The coordinates of the corresponding atomic models have been deposited in the Protein Data Bank under accession code 5fj8 (elongating Pol III) and 5fj9 (‘closed clamp’ apo Pol III), 5fja (‘open clamp’ apo Pol III).
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Acknowledgements
We thank Y. Sadian, H. Grötsch and R. Wetzel for help in Pol III purification. We are grateful to F. Thommen and M. Wahlers for set-up and maintenance of the high-performance computational environment of RELION. We acknowledge F. Schur, S. Fromm, C. Bertipaglia, M. Beck and G. Hofhaus for helpful advice regarding sample preparation and image processing. We also thank the ‘Fermentation et culture de microorganisms’ (IFR88, CNRS, Marseille). M.M.-M. and A.J.J. were supported by Marie-Sklodowska-Curie fellowships (FP7-PEOPLE-2011IEF301002, PIEF-GA-2012-331285). N.A.H. acknowledges support by the EMBL International PhD program, A.J.J. and J.K. by postdoctoral fellowships from the EMBL Interdisciplinary Postdoc Program (EIPOD) under Marie Curie COFUND actions (PCOFUND-GA-2008-229597) and C.W.M. by an ERC Advanced Grant (ERC-2013-AdG340964-POL1PIC).
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C.W.M. and C.S. initiated and supervised the project. N.A.H. established and performed Pol III experiments with input from C.W.M., S.G. and M.M.-M.; N.A.H., C.S. and W.J.H.H. established the grid freezing and acquisition parameters, N.A.H. and W.J.H.H. collected all cryo-EM data. N.A.H., M.M.-M. and A.J.J. built the initial model. N.A.H., A.J.J. and C.S. analysed cryo-EM data. J.K. performed homology modelling and alignments, A.J.J. established the refinement protocol. A.J.J. and N.A.H. optimized the refinement protocol and revised the models. N.A.H., A.J.J., S.G., M.M.-M., C.S. and C.W.M. wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Figure 1 Pol III processing pipeline, Fourier-shell correlation curves and local resolution assessment.
a, Exemplary micrograph of elongating Pol III. All micrographs were low-pass filtered for particle picking. b, General processing pipeline. The orange boxes display micrograph number and particles for elongating Pol III (left) and apo Pol III (right). The middle panel shows the general workflow that was followed for both data sets. For elongating Pol III (bottom left), a local classification step yielded one class with 49,543 particles (purple) that was subsequently refined and post-processed. For apo Pol III (bottom right), local classification diverged into two classes (purple) with 68,818 particles and 52,423 particles that were subsequently refined and post-processed. c, Fourier-shell correlation (FSC) and local resolution assessment with RESMAP55. All FSC calculations were performed with two independent half maps using RELION’s masking procedure53. The resolution for the elongating Pol III cryo-EM map (top panel) is 3.9 Å according to the FSC 0.143 criterion, indicated by the black dashed line. The two apo Pol III cryo-EM reconstructions have a resolution of 4.6 Å (closed clamp Pol III, middle panel) and 4.7 Å (open clamp Pol III, bottom panel) according to the FSC 0.143 criterion. Local resolution is displayed on the post-processed full maps (first image column on the right) and a cross-section representation (second image column on the right). In both apo Pol III reconstructions, the peripheral subcomplexes show a strong decay in resolution compared to the core. In the elongating Pol III reconstruction, the resolution is more uniformly distributed, indicating stabilization of peripheral subunits.
Extended Data Figure 2 Representative sections of the cryo-EM density for elongating Pol III.
a, Cross-section of elongating Pol III in ribbon and stick representation, embedded in the experimental density at 3.9 Å, displayed in dark blue. b, Section displaying the core subunits C160 (grey) and C128 (wheat) shown in stick and ribbon representation. The experimental density of the core (dark blue) is well defined and has been filtered at 3.5 Å resolution for display. c, Section of stalk subunits C25 (blue) and C17 (pink). The estimated local resolution in this part is lower compared to the core (Extended Data Table 2). In c, d and e, the cryo-EM density is shown at 3.9 Å resolution. d, Section showing subunits C53 (blue) and C37 (purple). e, Close-up view of C82-WH1 (brown), C82-WH2 (green) and C31 (yellow) interface.
Extended Data Figure 3 Comparison of electron microscopy densities with X-ray electron densities for shared subunits ABC23 (Rpb6) and ABC14.5 (Rpb8).
Top left shows Pol III in front view, a stretch in ABC23 (cyan) and ABC14.5 (green) is coloured. The red boxes indicate the regions that are enlarged in the neighbouring panels. Corresponding density is displayed in tungsten. Models of Pol II and Pol I at nominally higher resolution are available, but for better comparison models in a similar resolution range are shown. For the 2Fo − Fc electron density maps obtained by X-ray crystallography a threshold of 1σ was used for display. The top right shows three close-up views of the shared subunit ABC23 from elongating Pol III, Pol II (PDB 1wcm) and Pol I (PDB 4c3j). The bottom panels show 6 strands of shared subunit ABC14.5. Front view of the β-sheet and orthogonal views of individual strands in elongating Pol III, Pol II (PDB 1wcm) and Pol I (PDB 4c3j).
Extended Data Figure 4 Model validation and temperature factor distribution of atomic models.
a–c, FSC curves calculated between the refined atomic model and the half map used in refinement (FSCwork) are shown in blue, those calculated between the refined atomic model and the second half map not used for refinement (FSCtest) in red. Vertical lines mark the regular FSC 0.143 cutoff and the resolution target used in refinement as shown. Close agreement between FSCwork and FSCtest and the absence of a sharp drop beyond the refinement target resolution indicate that no overfitting took place. The respective FSC between the refined atomic model and the map obtained from 3D reconstruction using the entire data set (FSCref) is also shown (black). d, e, Atomic B-factor distributions mapped on ribbon representations of elongating and apo Pol III. The overall distribution and relative differences between core and peripheral subunits for the different models correlate well with the distribution of local resolution (Extended Data Fig. 1).
Extended Data Figure 5 Pol III-specific features of subunits C160 and C128 and comparison to the homologous Pol II and Pol I subunits.
a, Top (left) and front (right) view of Pol III, with subunits C160 and C128 displayed in ribbon representation and the other subunits in surface representation (grey). Coloured stretches highlight characteristic features denoted in b. b, Bar diagram shows the domain organization of Pol III C160. Arrows and corresponding numbers below the bar diagram indicate insertions and deletions of five or more residues in Pol III relative to Pol II subunit Rpb1 as indicated by structure-based alignment. Coloured regions are also shown in Pol III subunit C160 (lower panel, left) and in a. Lower panel middle and right show Pol II Rpb1 and Pol I A190 subunits, respectively. c, Same as in b for Pol III subunit C128. Insertions and deletions compared to Pol II subunit Rpb2 are displayed in the box diagram.
Extended Data Figure 6 Open and closed clamp conformation in Pol III compared to other RNA polymerases.
a, Top view of aligned elongating Pol III and apo Pol III (left panel, closed clamp; middle panel, open clamp) and both closed clamp and open clamp apo Pol III conformations (right panel). r.m.s.d. values (core-heterodimer:all) for elongating Pol III – apo Pol III (closed clamp) (0.43 Å3,490 Cα atoms aligned:0.43 Å4,813 Cα atoms aligned), elongating Pol III – apo Pol III (open clamp) (0.71 Å3,496 Cα atoms aligned:2.73 Å4,795 Cα atoms aligned) and both apo Pol III open and closed clamp (0.71 Å3,540 Cα atoms aligned:2.71 Å4,829 Cα atoms aligned) demonstrate the similarity between closed clamp apo Pol III and elongating Pol III conformations. b, Schematic representation of Pol III in top view showing the conformational changes of clamp head, heterotrimer and stalk. The closed clamp conformation (elongating Pol III and closed clamp apo Pol III) is displayed in red, the open clamp conformation (open clamp apo Pol III) in green. The DNA–RNA duplex is shown in blue, the core and heterodimer in grey. c, Front view on open and closed clamp conformations in other RNA polymerases. The closed clamp state (green) and open clamp state (red) is indicated for archaeal polymerase (left panel, PDB 4ayb and 4qiw), for Pol II (middle panel, PDB 1wcm and 1twf) and for Pol III (right panel). Green and red angles describe the cleft opening in the closed and open clamp conformations. Black arrows and corresponding values indicate the relative distance of the subunits between the two conformations. d, Front view of apo Pol III closed clamp (left panel), apo Pol III closed clamp vs apo Pol II (middle panel, Pol II (PDB 1wcm) in red) and apo Pol III closed clamp vs apo Pol I (right panel, Pol I (PDB 4c3i) in blue). The cleft opening is indicated by a dashed line and the Cα-Cα distance across the cleft (black for Pol III, red for Pol II and blue for Pol I). However, some of the observed differences in cleft width between Pol I, Pol II and Pol III might also reflect differences between conditions of cryo-EM and crystal structures as well as different packing contacts in the crystals.
Extended Data Figure 7 Pol III-specific subunits C82, C31 and C11.
a, Left panel: overall surface representation of Pol III with the C82–C34–C31 heterotrimer in ribbon representation. Right panels: two enlarged and orthogonal views of the region marked with a dotted black square. In subunit C82 WH4 inserts in the DNA binding cleft passing through a canyon in the clamp head. WH2 and WH3 extensions reach over the clamp head and are positioned in close proximity to downstream DNA. b, Ribbon model of Pol III fitted into the electron microscopy density of the open clamp apo Pol III filtered at 6 Å resolution. For C31, additional density is visible in the cavity between the stalk and the heterotrimer, as shown in the top right panel. The described densities are also present in the closed clamp apo Pol III and the elongating Pol III reconstructions. No attempts were made to fit atomic models into these densities. c, Electron microscopy density of the C11 TFIIS-like domain at 6 Å resolution as observed in the open clamp apo Pol III reconstruction. The left panel shows a side view of Pol III, the middle and right panels show close-ups at two different density thresholds.
Extended Data Figure 8 C53–C37 heterodimer and stalk subunits C25–C17.
a, Visualization of the photo-crosslinks between C53–C37 heterodimer and subunit C128. Pol III is shown in surface, C53–C37 and C11 in ribbon representation. In addition, the C128 lobe is shown in cartoon representation (small inset). Purple spheres on the lobe indicate residues that photo-crosslink to C37 (ref. 13), beige spheres on C37 indicate residues that photo-crosslink to C128 (ref. 13). The dashed line marks the tentative path of the non-template DNA strand. The experimental photo-crosslinks fit well to the cryo-EM structure. The C37 loop is disordered between Glu196 and Asn225, although photo-crosslinks indicate that this region is in close proximity to the lobe and the non-template DNA strand. b, Bottom view of Pol III in surface representation, with C53–C37 and C11 shown in ribbon representation. The black dotted square indicates the enlarged area in the centre of the image (small inset). The red density (shown at 4.5 Å) was not of sufficient quality to build an atomic model. However, photo-crosslinks from C37 and C128 to C53 (blue spheres on C37 ribbon and C128 surface mark crosslink positions)13 indicate that C53 N-terminal residues are located in this region. c, Stalk anchoring with C160 extensions. Top view (left panel) and bottom view (right panel) of the stalk subunits C17 (magenta), C25 (blue) and the C160 extensions (grey). Electron microscopy density corresponding to the C160 N- and C-terminal extensions is shown in tungsten blue. Individual entities and subunits are labelled.
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Hoffmann, N., Jakobi, A., Moreno-Morcillo, M. et al. Molecular structures of unbound and transcribing RNA polymerase III. Nature 528, 231–236 (2015). https://doi.org/10.1038/nature16143
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DOI: https://doi.org/10.1038/nature16143
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