Conformational flexibility of RNA polymerase III during transcriptional elongation

Abstract

RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 Å resolution and its elongation complex at 16.5 Å resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation.

Figure 1

Sub-nanometer structure of yeast Pol III. (A) Electron cryomicroscopy field of unstained Pol III particles (circle) in vitrified water. (B) Comparison of class averages (odd rows) and model reprojections (even rows) in different orientations. (C) Fourier shell correlation (FSC) function curve, showing a resolution of 9.9 Å when a cutoff value of FSC=0.5 is used. (D) Distribution of projection angles of the 3D reconstruction showing the approximate number of particles in each orientation. (E) Electron cryomicroscopy reconstruction of Pol III. Views and structural regions are named according to the Pol II structure (Cramer et al, 2001).

Figure 2

Structural differences between Pol III and II. (A) Fitting of the complete 12-subunit Pol II structure shown as ribbon model into the EM density (transparent surface). The fitting was performed in two rigid-body steps, first the 10-subunit core, then the stalk, as explained in the text. The structural elements showing the major differences (LC and IN stand for ‘low conservation' and ‘insertion') are labelled and numbered (see panel D for zoom). Subunit colouring is according to the legend on the right side. (B) Examples of regions where the temperature factor-sharpened EM map allows the identification of α-helices. (C) Fit of the common subunits Rpb5 and Rpb6 into the Pol III EM reconstruction. (D) Detailed views of some of the structural differences between the Pol III EM structure and the Pol II crystal structure. Zn²⁺ ions are depicted as green spheres. (E) Close-up view of the Pol III stalk. Left panel: position of the Pol II stalk with the 12-subunit crystal structure (1WCM) superimposed onto the fitted 10 subunits. Pol II subunits Rpb4 and Rpb7 are coloured pink and blue, respectively. Middle panel: independent fitting of the Pol II stalk into the Pol III electron cryomicroscopy reconstruction. The tip and OB domains in subunit Rpb7 (C25 orthologue) are indicated. Right panel: comparison of the Pol II stalk in the left panel with the independently fitted stalk structure.

Figure 3

Architecture of the Pol III-specific subcomplexes. (A) The additional density on the clamp element next to the stalk (orange) corresponds to the C31/C82/C31 heterotrimer, whereas the additional density next to the lobe element (blue) corresponds to the C37/C53 heterodimer. (B) Domain organization of subunits C82, C34, C37 and C53. The probability of a residue to fold into an α-helix or a β-strand is represented by a magenta and light-blue column, respectively. The propensity of a residue to be ordered is indicated below with a continuous black line (top ordered, bottom disorder). (C) SDS–PAGE analysis and gel-filtration profile of the purified C37/C53 dimerization domain. The elution volume corresponds to a globular mass of about 30 kDa, in agreement with that of the expressed domain (29 kDa). (D) Manual fitting of the C37/C53 dimerization domain and of five out of the six WH domains identified in subunits C82 and C34. The Rpb1 residue Thr69 important for the interaction of the Pol III core with the heterotrimer is shown in orange. The Rpb2 lobe and protrusion elements, identified as the docking region for C37/C53, are shown in magenta and pink, while the N-terminal domain of subunit Rpb9 equivalent to the N-terminal domain of Pol III subunit C11 is depicted in yellow.

Figure 4

Electron cryomicroscopy structure of the Pol III elongation complex. (A) Pol III elongation complex structure at 16.5 Å resolution. The Pol II elongation complex (Kettenberger et al, 2004) has been fitted into the EM density. Only the transcription bubble atomic structure is depicted in yellow. The insets show the largest, continuous segment of positive density in the difference map assigned to the transcription bubble, exiting RNA and the most important rearrangements of the Pol III-specific subcomplexes. (B) Difference map between Pol III (white semitransparent density) and its elongation complex with the positive and negative densities shown in red and blue, respectively. The most important structural rearrangements are indicated.

Figure 5

Pre-initiation complex models. (A) Domain organization of TFIIIB subunit Brf1. (B) Model of the closed PIC obtained by sequential superposition of the Pol II-TFIIB crystal structure (PDB code 3K7A) onto our Pol II core fit, then the TFIIB-TBP-DNA crystal structure (PDB code 1VOL) onto the obtained position of the B-core N-terminal cyclin fold, and finally the crystal structure of the Brf1-homology region II (residues 439–515) in complex with TBP and DNA (PDB code 1NGM) onto our previous positioning of TBP and DNA. An ideal B-form DNA was used to extend from the crystal structure, and the nucleotide positions −20, −10 and +1, +10 are indicated. The left panel orientation corresponds to the top view, rotated about 40° along a vertical axis. (C) Model of the open PIC obtained as before but, instead of adding a piece of straight DNA, we superposed the Pol II elongation complex (PDB code 1I6H) onto our Pol II core fit and, after removal of the RNA strand, connected the TATA-bound DNA to the elongating DNA with dashed lines.