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, 65 (Pt 2), 112-20

Structure-function Studies of the RNA Polymerase II Elongation Complex


Structure-function Studies of the RNA Polymerase II Elongation Complex

Florian Brueckner et al. Acta Crystallogr D Biol Crystallogr.


RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin alpha-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.


Figure 1
Figure 1
Structural overview of the complete 12-subunit RNA polymerase II elongation complex (Kettenberger et al., 2004 ▶). Two views are shown of a ribbon model of the protein subunits and nucleic acids, a side view (a) and a top view (b), related by a 90° rotation around a horizontal axis. The polymerase subunits Rpb1–Rpb12 are coloured according to the key shown below. Template DNA, nontemplate DNA and product RNA are shown in blue, cyan and red, respectively. P atoms are indicated as spheres and extrapolated B-form downstream DNA is coloured light pink. Eight zinc ions and the active-site magnesium ion are depicted as cyan spheres and a magenta sphere, respectively. This colour code is used throughout. Secondary-structure assignments for Pol II are according to Cramer et al. (2001 ▶) and Armache et al. (2005 ▶). This figure was adapted from Kettenberger et al. (2004 ▶) with modifications.
Figure 2
Figure 2
Structural details of the Pol II elongation complex. (a) Overview of the EC structure (Kettenberger et al., 2004 ▶). The view is as in Fig. 1 ▶(a). (b) Superposition of NTP-binding sites [red, insertion site (Westover et al., 2004a ; Wang et al., 2006 ▶); violet, entry site (Westover et al., 2004a ▶); pink, inactive pre-insertion-like state in which the triphosphate is too far from the catalytic metal ion A to allow incorporation (Kettenberger et al., 2004 ▶)]. (c) Functional Pol II surface elements in the EC highlighted in yellow. This figure was adapted from Cramer et al. (2008 ▶).
Figure 3
Figure 3
Schematic representation of the extended model for the nucleotide-addition cycle (NAC). The vertical dashed line indicates register +1. The steps where α-amanitin interferes with the NAC are indicated. For details, refer to the text. This figure was adapted from Brueckner & Cramer (2008 ▶) with modifications.
Figure 4
Figure 4
Structure of the α-amanitin-inhibited Pol II elongation complex. (a) Pre-translocation and post-translocation states of the EC. The nucleic acid scaffold used is depicted schematically with respect to the active-site metal ion A (magenta). The colour key is used throughout. (b) Overview of the α-amanitin-inhibited Pol II EC structure. The view is as in Fig. 1 ▶(a). α-Amanitin (stick model), nucleic acids (base in pre-templating position as a stick model), metal A, the bridge helix and the trigger loop (Leu1081 as a stick model) are highlighted using the colour key in (a). Part of the protein is omitted for clarity. (cd) Bromine anomalous difference Fourier maps (pink net) of the free EC (c) and the α-amanitin-inhibited EC (d). Br atoms are depicted as yellow spheres and their positions are indicated. The view is rotated by 90° around a vertical axis compared with (b). (e) The +1 DNA-template base adopts a pre-templating position. The initial unbiased F oF c difference map for the nucleic acids is shown around the +1 position and is contoured at 2.5σ. The +1 base in the pre-templating site is highlighted in violet. The view is rotated by 90° around a horizontal axis compared with (b). This figure was adapted from Brueckner & Cramer (2008 ▶).
Figure 5
Figure 5
Structures of Pol II (ab) and the Pol II EC (c) in complex with TFIIS. (a) Ribbon diagram of the Pol II–TFIIS complex backbone model (Kettenberger et al., 2003 ▶). The 12 subunits of Pol II are shown in silver. A pink sphere marks the location of the active-site metal ion A. Eight structural zinc ions in Pol II and one zinc ion in TFIIS are depicted as cyan spheres. The view is as in Fig. 1 ▶(a). (b) Binding of TFIIS to the jaw, crevice, funnel and pore. TFIIS is shown as a ribbon model on the molecular surface of Pol II. The view is from the bottom face, as indicated in (a). (c) TFIIS-induced RNA realignment (Kettenberger et al., 2004 ▶). Selected elements in the Pol II active centre that move upon TFIIS binding are shown. The bridge helix, DNA and RNA in the Pol II–bubble-RNA–TFIIS complex are shown in green, blue and red, respectively. The TFIIS hairpin is in orange, with the two acidic functionally essential and invariant residues in green. Nucleic acids in the Pol II–bubble-RNA complex structure after superposition of residues in the active-site aspartate loop or in switch 2 are shown in beige and grey, respectively. Switch 2 moves slightly upon TFIIS binding (Kettenberger et al., 2003 ▶), explaining the difference in the two superpositions. This figure was adapted from Kettenberger et al. (2003, 2004 ▶).
Figure 6
Figure 6
Mechanisms of DNA-damage recognition. (a) Structures of two different DNA dinucleotide lesions. The maximum lateral dimensions are indicated in green. (b) Structure of a cisplatin-damaged Pol II elongation complex (Damsma et al., 2007 ▶). Final 2F oF c electron-density map for the nucleic acids is shown (blue, contoured at 1.0σ). Anomalous difference Fourier map reveals the location of the Pt atom (magenta, contoured at 15σ). The cisplatin lesion is located outside of the active centre at positions +2/+3. This panel was adapted from Damsma et al. (2007 ▶). (c) Simplified mechanism of CPD DNA-damage recognition by Pol II. At the top, a schematic is shown that depicts the last few steps before Pol II stalling. At the bottom, nucleic acid structures in Pol II ECs containing a thymine–thymine CPD lesion before (left) and in the active site (right) are shown. DNA template, DNA nontemplate and RNA strands are shown in blue, cyan and red, respectively. The CPD is shown as a stick model in orange. The active-site magnesium ion (metal A) is depicted as a magenta sphere. This panel was adapted from Brueckner & Cramer (2007 ▶).

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    1. Armache, K.-J., Kettenberger, H. & Cramer, P. (2003). Proc. Natl Acad. Sci. USA, 100, 6964–6968. - PMC - PubMed
    1. Armache, K.-J., Mitterweger, S., Meinhart, A. & Cramer, P. (2005). J. Biol. Chem.280, 7131–7134. - PubMed
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