Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb
The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphory...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-08-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/48725 |
| _version_ | 1811200239141388288 |
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| author | Joshua E Mayfield Seema Irani Edwin E Escobar Zhao Zhang Nathaniel T Burkholder Michelle R Robinson M Rachel Mehaffey Sarah N Sipe Wanjie Yang Nicholas A Prescott Karan R Kathuria Zhijie Liu Jennifer S Brodbelt Yan Zhang |
| author_facet | Joshua E Mayfield Seema Irani Edwin E Escobar Zhao Zhang Nathaniel T Burkholder Michelle R Robinson M Rachel Mehaffey Sarah N Sipe Wanjie Yang Nicholas A Prescott Karan R Kathuria Zhijie Liu Jennifer S Brodbelt Yan Zhang |
| author_sort | Joshua E Mayfield |
| collection | DOAJ |
| description | The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes. |
| first_indexed | 2024-04-12T02:01:12Z |
| format | Article |
| id | doaj.art-53e0122eb0c34ddbb874f06e2b7e518b |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T02:01:12Z |
| publishDate | 2019-08-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-53e0122eb0c34ddbb874f06e2b7e518b2022-12-22T03:52:40ZengeLife Sciences Publications LtdeLife2050-084X2019-08-01810.7554/eLife.48725Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFbJoshua E Mayfield0Seema Irani1https://orcid.org/0000-0001-5159-3473Edwin E Escobar2https://orcid.org/0000-0002-0086-6264Zhao Zhang3Nathaniel T Burkholder4Michelle R Robinson5M Rachel Mehaffey6Sarah N Sipe7https://orcid.org/0000-0003-4554-7571Wanjie Yang8Nicholas A Prescott9https://orcid.org/0000-0002-0635-8906Karan R Kathuria10Zhijie Liu11Jennifer S Brodbelt12https://orcid.org/0000-0003-3207-0217Yan Zhang13https://orcid.org/0000-0002-9360-5388Department of Molecular Biosciences, University of Texas at Austin, Austin, United StatesDepartment of Chemical Engineering, University of Texas at Austin, Austin, United StatesDepartment of Chemistry, University of Texas at Austin, Austin, United StatesDepartment of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, United StatesDepartment of Molecular Biosciences, University of Texas at Austin, Austin, United StatesDepartment of Chemistry, University of Texas at Austin, Austin, United StatesDepartment of Chemistry, University of Texas at Austin, Austin, United StatesDepartment of Chemistry, University of Texas at Austin, Austin, United StatesDepartment of Molecular Biosciences, University of Texas at Austin, Austin, United StatesDepartment of Molecular Biosciences, University of Texas at Austin, Austin, United StatesDepartment of Molecular Biosciences, University of Texas at Austin, Austin, United StatesDepartment of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, United StatesDepartment of Chemistry, University of Texas at Austin, Austin, United StatesDepartment of Molecular Biosciences, University of Texas at Austin, Austin, United States; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, United StatesThe Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.https://elifesciences.org/articles/48725post-translational modificationP-TEFbtranscriptionpromoter-proximal pausingphosphorylationultraviolet photodissociation mass spectrometry |
| spellingShingle | Joshua E Mayfield Seema Irani Edwin E Escobar Zhao Zhang Nathaniel T Burkholder Michelle R Robinson M Rachel Mehaffey Sarah N Sipe Wanjie Yang Nicholas A Prescott Karan R Kathuria Zhijie Liu Jennifer S Brodbelt Yan Zhang Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb eLife post-translational modification P-TEFb transcription promoter-proximal pausing phosphorylation ultraviolet photodissociation mass spectrometry |
| title | Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb |
| title_full | Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb |
| title_fullStr | Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb |
| title_full_unstemmed | Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb |
| title_short | Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb |
| title_sort | tyr1 phosphorylation promotes phosphorylation of ser2 on the c terminal domain of eukaryotic rna polymerase ii by p tefb |
| topic | post-translational modification P-TEFb transcription promoter-proximal pausing phosphorylation ultraviolet photodissociation mass spectrometry |
| url | https://elifesciences.org/articles/48725 |
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