The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration

Abstract

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.

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 (β’ of bacterial RNAP, Rpo1’+Rpo1” 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 represent 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 Pyrococcus furiosus (Pfu) and Thermococcus kodakarensis (Tko) is highlighted.

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 color and labeled. Two sets of nomenclatures, one for traditional (in parenthesis) and the other based on the eukaryotic terminology, are shown. Some structural features are labeled. (b) Crystal structures of Rpo3/Rpo11 (left) and Rpo4/Rpo7 (right) complexes. Domains are labeled. Each subunit is colored as in a. (c) Surface representations of the archaeal and eukaryotic RNAPs. Each subunit is denoted by a unique color and labeled. Orthologous subunits are depicted by the same color. In a bottom panel, common subunits are shown in gray.

Figure 3. Opening and closing of the DNA binding clamp of RNAP

(a) The structure of Tko RNAP is superimposed on the structures of Sso RNAP (left, blue, PDB: 3HKZ) and yeast Pol II (right, green, PDB: 1WCM) using the enzyme active sites as references. (b) Cryo-EM density map of Pfu RNAP (light grey mesh, EMD-1711) is overlaid with the Tko RNAP structure (main body of RNAP, grey; clamp, dark cyan; Rpo4/Rpo7 stalk, yellow green; active site Mg, magenta sphere). The clamp of Pfu RNAP is indicated by a red arrow and in a closed-state. (c) Proposed motions of the clamp and stalk of archaeal RNAP. The open clamp (dark cyan) and stalk (yellow green) observed in the Tko RNAP structure are superimposed on the closed clamp and stalk (gray) in the Sso RNAP structure. Motions and angles of the clamp and stalk from the closed to the open conformations are indicated. (d) Schematic representation of archaeal RNAP/Pol II showing the mobility of the DNA-binding cleft and the stalk.

Figure 4. Structural differences between the Tko RNAP and yeast Pol II

(a) Schematic diagrams of domains and domain-like regions of the Tko RNAP based on the Pol II nomenclature. Inverted triangles indicate positions of 30 insertions found in yeast Pol II compared with Tko RNAP (black, insertions in eukaryotic RNAPs; red, Pol II-specific insertions; gray, unique in yeast Pol II). The binding sites of TFIIB, TFIIE, TFIIF, TFIIH and Mediator are indicated. Asterisks indicate positions of 4 insertions found in the Tko RNAP subunits. (b) The structure of yeast Pol II and the three-dimensional representation of the 30 insertions in a. Insertions are depicted by plain-surfaces with yellow boundaries (colors same as in a). Circles show approximate locations of disordered insertions at N- or C-termini of subunits (e.g., Pol II CTD) and their diameters represent their approximate lengths. 7 groups of insertions (I to VII) are indicated in blue rectangles.

Figure 5. The PIC model of Pol II

(a) Pol II, GTFs and DNA are depicted by surface, cartoon and cpk models, respectively. Insertions specific and non-specific to Pol II are indicated in red and pink, respectively. Each GTF is denoted by a unique color and labeled. (b) The Pol II and TFIIF interactions in the PIC. Pol II and TFIIF are depicted by surface and cartoon models, respectively. Rpb2 and Rpb9 are in white and yellow, respectively and all other subunits are in gray. Subunits and domains of TFIIF are labeled. Four insertions participated in the TFIIF binding are indicated. This PIC model is adapted from ref. .

Figure 6. Model of clamp conformation control through the stalk

(a) A nascent RNA, depicted as a dashed line, may stabilize the closed conformation of the clamp in the transcription elongation complex by the interaction with the stalk. (b) Binding of TFE/TFIIEα, shown as a gray ellipse, on the clamp and stalk may stabilize the open conformation of the clamp and stalk in the PIC.

Figure 7. Structure and sequence comparisons of Tko RNAP and yeast Pol II around binding sites of the Pol II-specific GTFs/Mediator

Structures of Pol II around the binding sites of TFIIF (a), TFIIH (b) and Mediator (c) and their counterparts in the Tko RNAP structure are shown. Insertions are colored and indicated in red and disordered regions are depicted as dashed lines. Amino acid sequence alignments around these regions are shown on their right (Tk, Tko; Ss, Sso; Sc, Sce; Hs, Homo sapiens). Amino acid residues cross-linked to TFIIF (a) and TFIIH (b) are indicated in red. Cys92 and Ala159 of Rpb3, which interact directly with Mediator, are indicated in red and the positions of i18 are indicated by a blue arrow on Tko RNAP and a blue circle on Pol II (c).

Figure 8. Pol II-specific insertions and TFIIH and Mediator binding interfaces on Pol II

(a) Pol II-specific insertions on the Pol II backbone model on the TFIIH binding interface. (b) Pol II-specific insertions on the Mediator binding interface. In both panels, insertions are shown as red plain-surfaces with yellow-green boundaries on the Pol II backbone model and key subunits and domains are indicated. Magnified views of these insertions are shown in boxes.