The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

ABSTRACT Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc −/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism.

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Structure of the p53/RNA polymerase II assembly

Communications Biology ◽

10.1038/s42003-021-01934-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Shu-Hao Liou ◽

Sameer K. Singh ◽

Robert H. Singer ◽

Robert A. Coleman ◽

Wei-Li Liu

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Conformational Changes ◽

P53 Protein ◽

Binding Activity ◽

Transactivation Domain ◽

Pol Ii ◽

Dna Binding Activity ◽

Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Is Required for Cotranscriptional Pre-mRNA Splicing and 3′-End Formation

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.8963-8969.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 8963-8969 ◽

Cited By ~ 78

Author(s):

Gregory Bird ◽

Diego A. R. Zorio ◽

David L. Bentley

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Kinase Inhibitors ◽

Mrna Splicing ◽

Pol Ii ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Ctd Phosphorylation

ABSTRACT We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3′-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or α-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3′-end processing is mediated by transcriptional coupling.

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RNA polymerase II clustering through CTD phase separation

10.1101/316372 ◽

2018 ◽

Cited By ~ 8

Author(s):

M. Boehning ◽

C. Dugast-Darzacq ◽

M. Rankovic ◽

A. S. Hansen ◽

T. Yu ◽

...

Keyword(s):

Phase Separation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Low Complexity ◽

Initiation Factor ◽

Published Data ◽

Pol Ii ◽

Intrinsically Disordered ◽

Ctd Phosphorylation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

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Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.1-15.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 1-15 ◽

Cited By ~ 25

Author(s):

Seiji Yamamoto ◽

Yoshinori Watanabe ◽

Peter J. van der Spek ◽

Tomomichi Watanabe ◽

Hiroyuki Fujimoto ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

In Vitro Transcription ◽

Pol Ii ◽

Transcription System ◽

Carboxy Terminal ◽

Transition To Elongation ◽

Ctd Phosphorylation

ABSTRACT The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

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Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

10.1101/652214 ◽

2019 ◽

Author(s):

Joshua E. Mayfield ◽

Seema Irani ◽

Edwin E. Escobar ◽

Zhao Zhang ◽

Nathanial T. Burkholder ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Elongation Factor ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Terminal Domain ◽

Ctd Phosphorylation

SummaryThe Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase II’s C-terminal domain (CTD) 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 a reduction of phosphorylated Ser2 and 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.

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Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

eLife ◽

10.7554/elife.48725 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 2

Author(s):

Joshua E Mayfield ◽

Seema Irani ◽

Edwin E Escobar ◽

Zhao Zhang ◽

Nathaniel T Burkholder ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Elongation Factor ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Terminal Domain ◽

Ctd Phosphorylation

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.

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hnRNP U Inhibits Carboxy-Terminal Domain Phosphorylation by TFIIH and Represses RNA Polymerase II Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.6833 ◽

1999 ◽

Vol 19 (10) ◽

pp. 6833-6844 ◽

Cited By ~ 46

Author(s):

Myung K. Kim ◽

Vera M. Nikodem

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Early Stage ◽

Pol Ii ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Hnrnp U ◽

Carboxy Terminal ◽

Ctd Phosphorylation

ABSTRACT This study describes a potential new function of hnRNP U as an RNA polymerase (Pol II) elongation inhibitor. We demonstrated that a subfraction of human hnRNP U is associated with the Pol II holoenzyme in vivo and as such recruited to the promoter as part of the preinitiation complex. hnRNP U, however, appears to dissociate from the Pol II complex at the early stage of transcription and is therefore absent from the elongating Pol II complex. When tested in the human immunodeficiency virus type 1 transcription system, hnRNP U inhibits elongation rather than initiation of transcription by Pol II. This inhibition requires the carboxy-terminal domain (CTD) of Pol II. We showed that hnRNP U can bind TFIIH in vivo under certain conditions and inhibit TFIIH-mediated CTD phosphorylation in vitro. We find that the middle domain of hnRNP U is sufficient to mediate its Pol II association and its inhibition of TFIIH-mediated phosphorylation and Pol II elongation. The abilities of hnRNP U to inhibit TFIIH-mediated CTD phosphorylation and its Pol II association are necessary for hnRNP U to mediate the repression of Pol II elongation. Based on these observations, we suggest that a subfraction of hnRNP U, as a component of the Pol II holoenzyme, may downregulate TFIIH-mediated CTD phosphorylation in the basal transcription machinery and repress Pol II elongation. With such functions, hnRNP U might provide one of the mechanisms by which the CTD is maintained in an unphosphorylated state in the Pol II holoenzyme.

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Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue

Biochemical Journal ◽

10.1042/bj20101471 ◽

2011 ◽

Vol 434 (3) ◽

pp. 435-444 ◽

Cited By ~ 27

Author(s):

Yong Zhang ◽

Mengmeng Zhang ◽

Yan Zhang

Keyword(s):

Rna Polymerase ◽

Transition State ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Complex Structure ◽

Protein Tyrosine Phosphatases ◽

Cysteine Residue ◽

Phosphoryl Transfer ◽

Terminal Domain ◽

Deep Groove

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.

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Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5562-5564.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5562-5564

Author(s):

S Buratowski ◽

P A Sharp

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Adenovirus Type ◽

Late Promoter ◽

Terminal Domain ◽

Late Transcription ◽

Major Late Promoter

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

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Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.00819-10 ◽

2010 ◽

Vol 30 (21) ◽

pp. 5180-5193 ◽

Cited By ~ 17

Author(s):

Alicia García ◽

Emanuel Rosonina ◽

James L. Manley ◽

Olga Calvo

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Large Subunit ◽

Terminal Domain ◽

Mrna Metabolism ◽

Genes Encoding ◽

The One ◽

Ctd Phosphorylation ◽

Transcription Cycle

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

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