Research ArticleBIOCHEMISTRY

Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

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Science Advances  07 Apr 2021:
Vol. 7, no. 15, eabd4420
DOI: 10.1126/sciadv.abd4420
  • Fig. 1 Cryo-EM structure of TFIIK.

    (A) Left: Cryo-EM map of the core yeast TFIIK at 3.5 Å shows clear density for each subunit: Kin28 (blue), Ccl1 (cyan), and Tfb3 (orange) with density colored by subunits indicated. Right: Structural model of TFIIK with subunits colored as indicated. Activation loop, ADP-AlF3, and phosphorylated Thr162 annotated. (B) Cryo-EM map and fit structural model of yeast TFIIK at 3.64-Å resolution, including HN3 and HN4 helices of Ccl1. (C to E) EM density with side chains of Kin28 (C), Ccl1 (D), and Tfb3 (E). (F) Schematic diagram of domains of TFIIK subunits. The Ring domain and helical domain of Tfb3 are not resolved in the EM map.

  • Fig. 2 TFIIH holo-enzyme cross-linking mass spectrometry.

    (A) Six hundred thirty-five nonredundant cross-links identified for holo-TFIIH containing TFIIK and core TFIIH components (Ssl2, Rad3, Tfb1, Tfb2, Ssl1, Tfb4, and Tfb5) as a network plot. Intrasubunit cross-links are shown in purple, and interprotein cross-links are shown in green. (B) Circle plot of TFIIK-specific cross-links. (C) Cross-links mapped on the structure of TFIIK, with all cross-links (blue) consistent with a cutoff of 25 Å, well below the 40-Å upper limit.

  • Fig. 3 Activation mechanism of TFIIK.

    (A) Structural model of TFIIK activation. Kin28 (blue), Ccl1 (cyan), Tfb3 (orange), and the activation loop (red) are colored. (B) EM density of the activation loop with important activating residues. The phosphate group on Thr162 (pT162) is apparent as in other activated CDKs (30). (C) Electrostatic potential map of activation loop and surrounding residues show a conserved basic patch surrounding pT162, suggesting a similar activation mechanism conserved through CDKs. (D) Activation of the Kin28 activation loop by the Tfb3 activation helix and the Kin28 KHYT motif. The Tfb3 activation helix makes direct contact with the activation loop via hydrogen bonding interaction between Tfb3 R304 and Kin28 backbone carbonyl and hydrophobic interaction between Tfb3 F296 and Kin28 P157. Tfb3 also binds the Kin28 KHYT motif (residues 180 to 183) by a hydrogen bonding networking including Tfb3 F291–Kin28 H181 and Tfb3 Y300–Kin28 T183. The KHYI motif also helps stabilize the activated activation loop by hydrogen boding interactions with Y182 and R128 and hydrophobic interaction between Y182 and L161. (E) Sequence alignment of Tfb3 activation loop (top) and Kin28 activation loop (bottom). Tfb3 activation helix is highly conserved from yeast to human. Kin28 activation loop is highly conserved though A156 is replaced with a serine/threonine in other eukaryotes for CAK regulation. (F) Structures of inactive human CDK7 (PDB: 1UA2) (pink, left), active yeast Kin28 (this study) (blue, middle), and active CDK2 (PDB: 1FIN) (purple, right). Inactive CDK7 has activation loop (red) covering the active site, while activated Kin28 and CDK2 moved the activation loop, which are stabilized by the Tfb3 activation helix and the CycA αN helix, respectively. The CycA αN helix is absent in Ccl1/cyclin H.

  • Fig. 4 Computational modeling of CTD peptide bound to Kin28.

    Rosetta model of one-repeat CTD peptide (PTSPSYS, the underlined phosphorylatable Ser5 at position +0 in the catalytic site) for Ser5 phosphorylation by TFIIK using human CDK2-substrate peptide complex (PDB: 3QHR) as a template. An ensemble of the five lowest-energy models is shown.

  • Fig. 5 TFIIK in the preinitiation-Mediator complex.

    (A) Probability distributions [Kin28 in blue, Ccl1 in cyan, the C-terminal 62 residues of Tfb3 (residues 259 to 320) in orange, and the linker region of Tfb3 (residues 146 to 267) in orange red] generated by Integrative Modeling based on cryo-EM map and XL-MS. Kin28, Ccl1, and Tfb3 are represented by coarse-grained beads, with each bead corresponding to 1 and 5 amino acid residues for structured and unstructured regions. (B) Placement of the structure of TFIIK in previous cryo-EM map of PIC-Mediator (EMDB: 3850). Mediator (green), TFIIH (yellow) and pol II (gray) represented in cylinders. Pol II CTD (purple, PDB: 4GWQ) is located in close proximity (<40 Å) to TFIIK. (C) Close-up view on connection of TFIIK to PIC through Tfb3. Tfb3 is anchored to TFIIH by the Ring domain connected to Rad3. Placement of TFIIK near Mediator Med6/Med8 would likely require the unfolding of helical linker domain of Tfb3 (Fig. 1F), schematically depicted by the orange dashed line. Previous studies (19, 20) identified cross-links between Mediator/pol II subunits and the Tfb3 helical domain (Tfb3 K192–Med8 K173, Tfb3 K226–Rpb4 K212, Tfb3 K192–Med11 K45, and Tfb3 K226–Med8 K173) and Kin28–Ccl1 binding domain (Ccl1 K273–Med11 K45). Residues of pol II/Mediator in black spheres formed cross-links with the helical domain of Tfb3, which is extended (orange dashed line) in our IMP (integrated modeling platform)-based modeling.

  • Fig. 6 Model of TFIIK and CTD in the preinitiation-Mediator complex.

    Proposed model of TFIIK in the PIC. The Mediator head module guides pol II CTD toward the active site of TFIIK. The C-terminal end of the 25-residue CTD segment (purple) bound to the Mediator head (39) coincides with the N-terminal end of the seven-residue CTD segment (purple) bound to TFIIK (this study). The two segments are connected by a ~14-residue linker (silhouette).

Supplementary Materials

  • Supplementary Materials

    Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

    Trevor van Eeuwen, Tao Li, Hee Jong Kim, Jose J. Gorbea Colón, Mitchell I. Parker, Roland L. Dunbrack, Benjamin A. Garcia, Kuang-Lei Tsai, Kenji Murakami

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