Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription

Abstract

The x-ray structure of complete RNA polymerase II from Saccharomyces cerevisiae has been determined, including a heterodimer of subunits Rpb4 and Rpb7 not present in previous "core" polymerase II structures. The heterodimer maintains the polymerase in the conformation of a transcribing complex, may bind RNA as it emerges from the enzyme, and is in a position to interact with general transcription factors and the Mediator of transcriptional regulation.

Fig. 1.

Complete, 12-subunit pol II electron density map. (A) Front view (as in refs. and 11) of SigmaA-weighted F_obs – F_calc electron density at 4.1-Å resolution (green) contoured at 3 σ, calculated from the initial placement of the pol II model (dark gray). The initial placement of archaeal RpoF (Rpb4 homolog) is shown in red, and placement of archaeal RpoE (Rpb7 homolog) in shown in blue. (B) Electron density map at 4.1-Å resolution (yellow) contoured at 1 σ, calculated by using observed amplitudes (F_obs) and phases after density modification. Superimposed is the final C^α Rpb4 (red) and Rpb7 (blue) model. The figure was generated by using O and POV-RAY (19).

Fig. 2.

Backbone model of complete, 12-subunit pol II. Shown is a ribbon representation of the complete pol II structure (top and back views as in refs. and 11). Rpb1 is gray, Rpb2 is bronze, Rpb4 is red, Rpb6 is green, the N-terminal half of Rpb7 that contains the RNP domain is dark blue, the C-terminal half of Rpb7 that contains the OB fold is light blue, and the remaining subunits are black. The locations of the clamp, the C-terminal repeat domain (CTD), and the previously proposed RNA exit groove 1 (pink dashed line) are indicated. The figure was generated with SWISS-PDB VIEWER and POV-RAY (40).

Fig. 3.

Relationship of complete pol II x-ray structure to EM structures of complete pol II (A) and Mediator–pol II complex (B). As this complex was prepared from exponentially growing yeast, it would have been largely deficient in Rpb4/Rpb7, accounting for the lack of density in this region of the EM map. The core pol II model is blue in A and yellow in B. Rpb4 is red and Rpb7 is dark blue. The figure was generated by using O and POV-RAY (19).