Molecular basis of transcription initiation in Archaea

Abstract

Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.

Keywords: RNAP; archaea; cryo-electron microscopy; initiation; molecular fitting; transcription.

Figure 1

Cryo-EM analysis of apo-RNAP. (A) Negative staining EM and selected class-averages of the P. furiosus apo-RNAP. Each class-average is composed of about 10–30 raw images and was aligned against an initial 3-D reference to define its orientation parameters. (B) cryo-EM micrograph and selected class-averages, illustrated with reversed contrast. (C) Fitting of the S. solfataricus apo-RNAP crystal structure (PDB 2PMZ) into the cryo-EM refined model. The crystal structure was manually fitted in chIMeRa and the fit was refined with a least-square minimization algorithm (see Materials and Methods). Scale bar = 65 nm in (A and B); 5 nm in (C).

Figure 2

Functional analysis and purification of P. furiosus PIC (A, left) silver stain gel of purified RNAP from P. furiosus and reconstituted transcription. RNAP, TBP and/or TFB were added as shown. Asterisk, minor cryptic initiation site in the absence of TBP/TFB; Arrow, correct initiation site from the T6 promoter. (B) Coomassie-stained gel for PIC cross-linking. The letters B through P denote RNAP subunits; TBP and TFB are also indicated. (C) Elution profile for the cross-linked PIC purified with size-exclusion chromatography on a 24 ml Superdex 200 column. Time = 7 min is the column dead volume; the wavelength was set to 280 nm.

Figure 3

EM analysis of the P. furiosus PIC. (A) Negatively-stained micrograph and selected class-averages of the PIC (RNAP /TBP/TFB/DNA complex). Each class-average is composed of about 10–30 raw images and was aligned against an initial RNAP 3-D reference to define its orientation parameters. (B) PIC reconstruction (top) compared to apo-RNAP (bottom) in two different orientations. Extra density within PIC structure is indicated by red arrows. (C) Overlay of the RNAP cryo-EM structure (from Fig. 1) or the X-ray structure onto the EM density map of the PIC. A red star highlights extra density.

Figure 4

Localization of TBP/TFB within the P. furiosus PIC. (A) Back-view of the PIC EM structure (grey mesh) with fitted DNA/TBP/TFB crystal structure (PDB ID 1AIS). (B) Same as (A), but the fit was improved by rotating the DNA/TBP/TFB complex about 30 degrees. (C) Proposed model of Archaeal PIC: top-view of the PIC EM density (grey) according to the improved fit shown in (B). (D) High-resolution model proposed for the Archaeal PIC (RNAP from PDB ID 2PMZ; DNA/TBP/TFIIB from PDB ID 3K1F). Scale bar = 5 nm in (A and B) and 10 nm in (C and D).

Figure 5

A conserved core architecture within archaeal and eukaryotic PIC? High-resolution model for (A) archaeal PIC. and (B) Yeast RNAP II/TFIIB complex from PDB ID 3K1F. (C) Subunits composition of Archaea RNAP and Eukaryotic RNAP II. Scale bar = 10 nm.