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

Irani, Seema; Sipe, Sarah N.; Yang, Wanjie; Burkholder, Nathaniel T.; Lin, Brian; Sim, Kelly; Matthews, Wendy L.; Brodbelt, Jennifer S.; Zhang, Yan

doi:10.1074/JBC.RA119.007697

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

Journal Article · Wed Apr 10 00:00:00 EDT 2019 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/JBC.RA119.007697· OSTI ID:1604191

Irani, Seema ^[1]; Sipe, Sarah N. ^[1]; Yang, Wanjie ^[1]; Burkholder, Nathaniel T. ^[1]; Lin, Brian ^[1]; Sim, Kelly ^[1]; Matthews, Wendy L. ^[1]; Brodbelt, Jennifer S. ^[1];

^[1]

Univ. of Texas, Austin, TX (United States)

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.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institutes of Health (NIH); Welch Foundation

Grant/Contract Number:: R01 GM104896; R01 GM125882; F-1778

OSTI ID:: 1604191

Journal Information:: Journal of Biological Chemistry, Vol. 294, Issue 21; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 5 works

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Related Subjects

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

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

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