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Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription

Journal Article · · Journal of Biological Chemistry
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. Univ. of Texas, Austin, TX (United States)
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The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II–mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, in this work we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2. The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight β-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.
Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
National Institutes of Health (NIH); Welch Foundation
OSTI ID:
1604191
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 21 Vol. 294; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Figures / Tables (5)

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
CTD code
DNA transcription
RNA polymerase II
X-ray crystallography
intrinsically disordered protein
mass spectrometry (MS)
phosphatase
phosphoryl motif
proline isomerization
tyrosine phosphorylation