The X-ray crystal structure of RNA polymerase from Archaea

Abstract

The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 A resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 A) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

Figure 1

Three-dimensional structure of the archaeal RNAP. a, 3.4 Å resolution crystal structure of the S. solfataricus RNAP. Each subunit is denoted by a unique color (see surface representations in Fig. 2 for color-code and subunit-subunit interaction). The disordered clamp head domain is indicated as a dotted line. b, 1.76 Å resolution crystal structure of the D/L subcomplex (red, D subunit; yellow, L subunit). Domain organization is shown. c, Close-up view of the 4Fe-4S cluster binding domain (residues 167–222 of the D subunit). Electron density calculated using the Fe anomalous signal is shown in black mesh (sigma cutoff =5).

Figure 2

Cellular RNAP structures from three domains of life. Surface representation of multi-subunit cellular RNAP structures from Bacteria (left, Thermus aquaticus core enzyme30), Archaea (center, S. solfataricus) and Eukarya (right, S. cerevisiae RNAPII13). Each subunit is denoted by a unique color and labeled. Orthologous subunits are depicted by the same color.

Figure 3

Structures around the foot domains from three domains of life. a, Ribbon model of the archaeal RNAP (color coding of each subunits are indicated). Various domains and motifs are labeled. The junction between A' and A” is positioned at foot domain. Subunits H and K associate around the base of the foot. b, Ribbon model of the eukaryotic RNAPII. The same four α-helix architecture, which is found in the archaeal RNAP, is conserved in the center of the RNAPII foot domain. Rpb5 and Rpb6 of also associate around the foot domain. c, Ribbon model of the bacterial RNAP. The bacterial foot domain has a completely different architecture. The right side of the bacterial foot domain associates with the bacterial specific insertion of the β' subunit. In addition, the ω tail wraps around the C-terminus of the β' subunit.

Figure 4

The Fe-S cluster may play a structural role in D subunit folding. a, Wild-type or variant D subunits were overexpressed in E. coli with the L subunit. Partially purified proteins were analyzed by gel-filtration column chromatography (top: wild-type, bottom: variant). Elution of marker proteins are also shown (1: γ-globulin/158 kDa; 2: ovalbumin/44 kDa; 3: myoglobin/17 kDa). The wild-type D and L subunits formed an heterodimer, and it was eluted ~62 ml (a, top panel: peak I, mean MW: ~30 kDa, MW of the subcomplex is 40.6 kDa), which was confirmed by Coomassie stained SDS-PAGE (b). The D subunit variant was eluted ~43 ml (a, bottom panel: peak II, mean MW: ~150 kDa) without L subunit that was verified by Western blot analysis using antibody against S. solfataricus subcomplex D/L (c).