Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
1195–1201
Year published:
DOI:
doi:10.1038/nsmb.1893
Received
Accepted
Published online

Abstract

Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′ end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.

At a glance

Figures

  1. Sequence alignment of CIDs and diheptad CTD phosphopeptides used in this study.
    Figure 1: Sequence alignment of CIDs and diheptad CTD phosphopeptides used in this study.

    (a) Sequence alignment of yeast (Sc) Rtt103, Pcf11, Nrd1 and human (Hs) SCAF8 and SCAF4 CIDs. Red, identical residues; purple, conserved amino acids. Secondary structure of Rtt103 is above the sequence. Blue dots identify Rtt103 residues that bind to the CTD in our structure. Cyan box marks positions of Arg108 and Asn107 in Rtt103 and Pcf11, respectively, and the corresponding residues in the Nrd1 and the SCAF CIDs. (b) Schematic of diheptad CTD peptides used in this study. Sequence at top; sites of serine phosphorylation, orange circles.

  2. Binding of Rtt103-CID and Pcf11-CID to diheptad CTD phosphopeptides monitored by fluorescence anisotropy and NMR.
    Figure 2: Binding of Rtt103-CID and Pcf11-CID to diheptad CTD phosphopeptides monitored by fluorescence anisotropy and NMR.

    (a) Rtt103-CID titrated into 2 μM FAM–labeled Ser2P-CTD diheptad repeat peptide (•), which competes against 25 μM Ser2P-CTD diheptad repeat peptides (), 50 μM Ser5P-CTD diheptad repeat peptides (), and 50 μM Ser2P and Ser5P-CTD diheptad repeat peptides () for binding. (b) Titration of Pcf11, as described in a except that 300 μM of unlabeled peptides were used. (c) Superposition of 1H-15N HSQC spectra of Rtt103 at several points in the titration with the doubly Ser2-phosphorylated CTD peptide showing changes in chemical shift on peptide binding. (d) HSQC perturbations were used to calculate binding affinities on a residue-by-residue basis for all amino acids showing substantial chemical shift changes on peptide binding by plotting the change in chemical shift versus the peptide concentration and by fitting the data to equation (1) in Supplementary Methods.

  3. Structure of Rtt103-CID and recognition of the Ser2P CTD.
    Figure 3: Structure of Rtt103-CID and recognition of the Ser2P CTD.

    (a) Ensemble of the 20 lowest-energy structures of the free Rtt103-CID as determined by NMR. (b) Superposition of the four existing CID structures: Rtt103, green, this study; Pcf11, cyan24; SCAF8, pink22 and Nrd1, gray20. (c) Structure of Rtt103 bound to the Ser2P (S2b-P) CTD (yellow). (d) Close-up of recognition of phosphorylated CTD by Rtt103 highlighting residues that directly contact the peptide. This and all subsequent structural figures were generated with PyMOL36.

  4. CTD specificity can be altered by a single-residue change.
    Figure 4: CTD specificity can be altered by a single-residue change.

    (a) Arg108 in Rtt103 makes several direct contacts to the CTD Ser2 phosphate (S2b-P); it is within hydrogen bonding distance of both the phosphoserine (S2bP) and the threonine (T4b) in the CTD (dotted black lines). Several other hydrogen bonds (dotted black lines) are characteristic of the β-turn structure. (b) Affinities (± s.d.) for wild-type and mutant Rtt103 and Pcf11 CIDs determined by anisotropy and NMR, respectively, with the Ser2abP diheptad CTD peptide. (c) Recruitment of C-terminally TAP-tagged Rtt103 and Rtt103 R108N to the highly transcribed PMA1 was monitored by ChIP assay. Immunoprecipitated DNA was amplified with PMA1 primers as diagrammed at bottom. Top, PMA1-specific; common lower band (star), internal background control from a nontranscribed region on chromosome VI. Middle, quantification of ChIP data, expressed as fold enrichment over background. (d) Recruitment of TAP-tagged wild-type Pcf11 and Pcf11 N107R to PMA1 was also monitored by ChIP assay; symbols as described in c.

  5. Cooperative binding of CID to phosphorylated CTD phosphoisoforms.
    Figure 5: Cooperative binding of CID to phosphorylated CTD phosphoisoforms.

    (a) Schematic of extended CTD phosphopeptides composed of four CTD heptad peptide repeats; phosphorylated serines (pSer), orange circles. (b) Chemical shift perturbations (CSP) of Rtt103 with the four-heptad repeat peptide phosphorylated at Ser2. Resonances showing increased CSPs with the four-repeat peptides, purple; resonances showing changes with both two- and four-repeat peptides, blue. (c) Residues showing chemical shift perturbations due to protein-protein interactions (purple) are primarily localized near the C terminus of helix 7; coloring as in b. (d,e) ChIP assays for the HA-tagged protein and Rpb3 from cells expressing wild-type HA-tagged Rtt103 (YSB2537) or Rtt103 E115R (YSB2538), in d; and wild-type HA-tagged Pcf11 (YSB2535) or Pcf11 D117A (YSB2536), in e. WT, wild type. Results are shown only for the poly(A) region, where Rtt103 or Pcf11 association is strongest (primer set 7 for PMA1 and 3 for ADH1). Error bars, s.d. from three repeats of the experiment.

  6. Cooperative model of Pcf11 and Rtt103 recruitment.
    Figure 6: Cooperative model of Pcf11 and Rtt103 recruitment.

    The density of Ser2P heptad repeats affects the location of transcription termination through cooperative recruitment of Pcf11 and Rtt103. At the poly(A) site, the density of Ser2P is highest15, allowing efficient recruitment of Pcf11 and Rtt103.

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References

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Author information

Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, Washington, USA.

    • Bradley M Lunde &
    • Gabriele Varani
  2. Biomolecular Structure and Design Program, University of Washington, Seattle, Washington, USA.

    • Bradley M Lunde
  3. Department of Chemistry, University of Washington, Seattle, Washington, USA.

    • Steve L Reichow,
    • Thomas C Leeper,
    • Fan Yang &
    • Gabriele Varani
  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Minkyu Kim,
    • Hyunsuk Suh &
    • Stephen Buratowski
  5. Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea.

    • Minkyu Kim
  6. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, Germany.

    • Hannes Mutschler &
    • Anton Meinhart

Contributions

B.M.L. carried out all NMR titration and fluorescence anisotropy experiments with the four-heptad repeat CTD peptides. Diheptad repeat fluorescence anisotropy experiments were done by H.M. H.M. wrote the scripts for fitting the fluorescence anisotropy measurements. B.M.L. and H.M. analyzed the fluorescence anisotropy data. Structure determination of Rtt103-CID was done by S.L.R. Rtt103-CID bound to the Ser2P CTD was determined by B.M.L. Isotope-filtered NMR experiments were collected by T.C.L. All mutants were made by B.M.L., while F.Y. collected and analyzed NMR relaxation experiments. A.M. made the Pcf11-CID and Rtt103-CID constructs and developed the expression and purification conditions. S.B., M.K. and H.S. designed the in vivo ChIP experiments, and M.K. and H.S. constructed the strains and carried out the assays. B.M.L., S.L.R., H.S., S.B., A.M. and G.V. wrote the paper.

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