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The X-Ray Crystal Structure of the Euryarchaeal RNA Polymerase in an Open-Clamp Configuration

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The X-Ray Crystal Structure of the Euryarchaeal RNA Polymerase in an Open-Clamp Configuration ARTICLE Received 6 Jun 2014 | Accepted 2 Sep 2014 | Published 14 Oct 2014 DOI: 10.1038/ncomms6132 The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration Sung-Hoon Jun1, Akira Hirata1,2, Tamotsu Kanai3, Thomas J. Santangelo4, Tadayuki Imanaka5 & Katsuhiko S. Murakami1 The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase II (Pol II) system. Archaeal RNA polymerase (RNAP) and Pol II evolved from a common ancestral structure and the euryarchaeal RNAP is the simplest member of the extant archaeal–eukaryotic RNAP family. Here we report the first crystal structure of euryarchaeal RNAP from Thermococcus kodakarensis (Tko). This structure reveals that the clamp domain is able to swing away from the main body of RNAP in the presence of the Rpo4/Rpo7 stalk by coordinated movements of these domains. More detailed structure–function analysis of yeast Pol II and Tko RNAP identifies structural additions to Pol II that correspond to the binding sites of Pol II-specific general transcription factors including TFIIF, TFIIH and Mediator. Such comparisons provide a framework for dissecting interactions between RNAP and these factors during formation of the pre-initiation complex. 1 Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. 2 Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790- 8577, Japan. 3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510 Japan. 4 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA. 5 Department of Biotechnology, Ritsumeikan University, Noji-Higashi, Kusatsu 525-8577, Japan. Correspondence and requests for materials should be addressed to K.S.M. (email: [email protected]) or to A.H. (email: [email protected]). NATURE COMMUNICATIONS | 5:5132 | DOI: 10.1038/ncomms6132 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6132 rchaeal proteins associated with genome maintenance and Rpo4/Rpo7 stalk. Structure-guided sequence alignment between gene expression have extensive functional and structural Tko RNAP and yeast Pol II postulates how retained insertions Asimilarities with their eukaryotic counterparts1,2. This and modifications to Pol II during RNAP evolution have been congruence is especially true for the archaeal transcription utilized to establish interactions with Pol II-specific GTFs and machinery, and there is a striking structural similarity between Mediator. Our structure–function analysis provides insight archaeal and eukaryotic RNA polymerases (RNAPs)3–5. regarding the evolution of multisubunit RNAPs with their Comparing the pre-initiation complex (PIC) formation of the binding factors and also serves as a guide for studying the archaeal and three eukaryotic transcription systems (Pol I, II and physical interactions between Pol II and transcription regulators. III) revealed that all RNAPs use a core subset of structurally and functionally related transcription factors to initiate promoter- Results dependent transcription6. All factors are auxiliary for the archaeal Tko RNAP purification and crystallization. The phylogenetic and Pol II transcription systems; however, some factors are bona analysis of the largest subunit of cellular RNAPs indicates fide RNAP subunits for the Pol I and Pol III transcription that among Euryarchaeota, Thermococcales including Tko is the systems. Archaeal RNAP is most closely related to Pol II in closest forms of RNAP to the common ancestor of the archaeal– subunit composition, and their requirements for general eukaryotic RNAP family (Fig. 1). Therefore, Tko RNAP can be transcription factors (GTFs) exactly match a subset of GTFs used as an ideal reference to analyse the structure and evolution required for the activities of Pol II. Archaeal RNAP requires only of archaeal–eukaryotic RNAP family15. Tko RNAP purified two monomeric GTFs—TBP and TFB—for PIC formation and directly from cells contains substoichiometric amounts of transcription in vitro, although a third monomeric GTF—TFE— TFE16, and this heterogeneity likely precluded crystallization can assist PIC formation in vitro and appears essential factor attempts. Tko RNAP purified from a Drpo4 strain yields an in vivo1,3,7,8. PIC formation with Pol II requires a more complex enzyme that lacks Rpo4, Rpo7 and TFE16. Introduction of set of GTFs, with minimally six GTFs (TFIIA, TFIIB, TFIID/TBP, recombinant Rpo4 and Rpo7 into this TFE-free RNAP reformed TFIIE, TFIIF and TFIIH) and the Mediator complex is also the full 11-subunit enzyme (Supplementary Fig. 1) that could be required for promoter-specific transcription7,9,10. crystallized successfully. The structure was determined by Archaea consists of two major phyla, Euryarchaeota and molecular replacement using the Sulfolobus solfataricus RNAP Crenarchaeota, and phylogenetic analyses of the essential structure (PDB ID 3HKZ)1 as a search model. We also solved the components in DNA replication, transcription and translation high-resolution structures of heterodimers formed by Tko RNAP suggested that Euryarchaeota have retained a set of features that subunits including Rpo3/Rpo11 (1.6 Å) and Rpo4/Rpo7 (2.3 Å; more likely represent the ancestral form present in the last Supplementary Table 2), and replacement with these structures common ancestor of eukaryotes and archaea11,12. Euryarchaeal allowed refinement of the final structure of Tko RNAP at 3.5 Å RNAP is composed of 11 subunits and all subunits are conserved resolution with high quality (Supplementary Fig. 2 and in the archaeal–eukaryotic RNAP family (Supplementary Supplementary Table 2). Table 1), whereas crenarchaeal RNAP contains two additional subunits Rpo8 and Rpo13 (refs 13,14). Although structural and functional similarities between archaeal and Pol II transcription The Tko RNAP structure. The overall shape of Tko RNAP machineries have been known for decades, precise comparison of resembles that of the crenarchaeal RNAP and eukaryotic Pol I these RNAPs generate new insights about structural motifs of and Pol II (Fig. 2). All subunits of Tko RNAP are conserved in RNAP that participate in the assembly of the PIC and archaeal–eukaryotic RNAPs supporting that the Tko RNAP transcription regulation. structure represents the closest form to their common ancestor We report the crystal structure of Thermococcus kodakarensis (Fig. 2c). Superposition of the Tko RNAP structure with the Sso (Tko) RNAP at 3.5 Å resolution, which reveals the molecular RNAP and yeast Pol II structures, both captured in the closed- details of the open-clamp state of the RNAP in the presence of the clamp conformation17,18, reveals that the Tko RNAP clamp is in 100 Methanosarcina acetivorans 100 Methanococcoides burtonii Haloarcula vallismortis 81 100 Halococcus morrhuae Euryarchaeota 91 Methanococcus maripaludis Pyrococcus furiosus 90 100 Thermococcus kodakarensis 100 Sulfolobus solfataricus Common ancestor of Sulfolobus acidocaldarius archaeal–eukaryal RNAP 100 99 Aeropyrum pernix Crenarchaeota 68 Pyrobaculum aerophilum 100 Pyrobaculum calidifontis Saccharomyces cerevisiae Eukaryote 100 Homo sapiens Escherichia coli Thermus thermophilus Bacteria 100 Thermus aquaticus 0.5 Figure 1 | Phylogenetic analysis of the largest subunit of RNAP in bacteria, euryarchaeota, crenarchaeota and eukaryotes. Maximum-likelihood phylogenetic tree made with the largest subunit of RNAP (b0 of bacterial RNAP, Rpo10 þ Rpo100 of archaeal RNAP, and Rpb1 of eukaryotic RNAP II) rooted with bacterial sequences. Bootstrap support based on 500 replicates is shown at each node. Scale bar represents the average number of substitutions per residues. The position of common ancestor of archaeal–eukaryal RNAP is indicated in red. An order of Thermococcales including Pfu and Tko is highlighted. 2 NATURE COMMUNICATIONS | 5:5132 | DOI: 10.1038/ncomms6132 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6132 ARTICLE Jaw/ Lobe/ lobe protrusion Rpo3/11 Rpo3/11 Cleft Cleft 180° Clamp Clamp Rpo4/7 Rpo4/7 (stalk) (stalk) Rpo1′, 1″ (RpoA′, A″) Rpo7 (RpoE) Rpo2 (RpoB) Rpo10 (RpoN) Rpo3 (RpoD) Rpo11 (RpoL) Rpo4 (RpoF) Rpo12 (RpoP) Rpo5 (RpoH) Mg Rpo6 (RpoK) Zn Tip loop Domain 2 Domain 3 C Tip-associated domain HRDC domain OB domain Dimerization domains Euryarchaeal RNAP Crenarchaeal RNAP Eukaryotic Pol II Eukaryotic Pol I (T. kodakarensis) (S. solfataricus) (S. cerevisiae) (S. cerevisiae) A34.5 9 A49 10 10 10 2 2 A135 10 2 3 11 12 3 3 11 12 8 AC19 11 12 12 AC40 ′ ′ 1 1 8 1 6 6 6 A190 6 74 7 4 7 4 A43 A14 180° 180° 180° A49 9 A34.5 AC40 3 3 3 A12.2 ″ 8 ″ 8 1 2 1 2 2 8 5 A135 11 11 5 5 5 13 ′ 1 6 1′ 6 1 6 6 A190 7 4 4 4 7 7 A14 A43 A49 9 A34.5 A12.2 8 8 8 13 Figure 2 | Crystal structures of the Tko RNAP and other members of archaeal–eukaryotic RNAP family. (a) Tko RNAP structure in a ribbon model along with a transparent molecular surface. Each subunit is denoted by a unique colour and was labelled. Two sets of nomenclatures, one for traditional (in parenthesis) and the other based on the eukaryotic terminology, are shown. Some structural features are labelled. (b) Crystal structures of Rpo3/Rpo11 (left)
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