Molecular mechanism of recruitment of TFIIF- associating RNA polymerase C-terminal domain phosphatase (FCP1) by transcription factor IIF

Abstract

After mRNA transcription termination in eukaryotes, the hyperphosphorylated form of RNA polymerase II (pol II0) must be recycled by TFIIF-associating C-terminal domain phosphatase (FCP1), the phosphatase responsible for dephosphorylating the C-terminal domain of the largest polymerase subunit. Transcription factor (TF)-IIF stimulates the activity of FCP1, and the RNA polymerase II-associating protein 74 subunit of TFIIF forms a complex with FCP1 in both human and yeast. Here, we report a cocrystal structure of the winged-helix domain of human RNA polymerase II-associating protein 74 bound to the alpha-helical C terminus of human FCP1 (residues 944-961). These results illustrate the molecular mechanism by which TFIIF efficiently recruits FCP1 to the pol II transcription machinery for recycling of the polymerase.

Figure 1

FCP1 domain organization, sequence, and RAP74 binding. (a) Domain organization of human FCP1 (red, FCPH catalytic portion; green, BRCT subdomain; orange, TFIIF RAP74 interacting region). (b) Sequence alignment of C-terminal regions of FCP1 from Homo sapiens, X. laevis, Drosophila melanogaster, Caenorhabditis elegans, S. cerevisiae, and S. pombe with α-helical (rectangle), random coil (solid lines), and disordered (dotted line) regions indicated. Amino acids making significant contacts with hRAP74cc are shaded yellow. [Note: residue numbering in this manuscript corresponds to the full-length human sequence, GenBank accession no. AAD42088 (ref. 9).] (c) hRAP74cc binding to GST-hFCP1pep (941–961). Lane designations: M, size markers; N, negative control (GST plus 16 unrelated residues); P, positive control (GST with WT hFCP1pep: GSTpep); 4–17, alanine-scanning mutants with residue numbering keyed to b.

Figure 2

Structure of hRAP74cc-hFCP1pep complex. ribbons (34) drawing of hRAP74cc (blue) bound to hFCP1pep (orange) with labeled N and C termini and secondary structural elements. Zinc (*, crystallographically identical divalent cation), sulfate ions, and zinc-binding residues are shown as color-coded ball-and-stick figures: S, green; C, yellow; O, red; N, blue; and Zn²⁺, magenta.

Figure 3

FCP1 binding to TFIIF. (a) grasp (35) representation of the molecular surface of hRAP74cc, color coded for electrostatic potential (red <−15k_BT, blue >+15k_BT, where k_B is the Boltzmann constant and T is temperature in Kelvins, calculated in the absence of peptide). hFCP1pep is depicted as a ball-and-stick figure. The view and color coding are as in Fig. 2. (b) hRAP74cc-hFCP1pep complex viewed close to the cylindrical axis of the peptide α-helix (C → N, ≈90^o rotation about horizontal from a), showing hRAP74cc surface residues (gray) and hFCP1pep (atomic stick figure, color coded as in a). C terminus is labeled, and hydrogen bonds and salt-bridges are denoted with broken magenta lines.