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

2011 ◽  
Vol 434 (3) ◽  
pp. 435-444 ◽  
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.

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

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.

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

2007 ◽  
Vol 27 (6) ◽  
pp. 2059-2073 ◽  
Author(s):  
Victoria H. Cowling ◽  
Michael D. Cole
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Target Genes ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Terminal Domain ◽  
Ctd Phosphorylation

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.

scholarly journals A three-step pathway of transcription initiation leading to promoter clearance at an activation RNA polymerase II promoter.

1996 ◽  
Vol 16 (4) ◽  
pp. 1614-1621 ◽  
Author(s):  
Y Jiang ◽  
M Yan ◽  
J D Gralla
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Potential Effect ◽  
Multiple Roles ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Step Model ◽  
Dna Strands

The progress of transcription bubbles during inhibition in vitro was followed in order to learn how RNA polymerase II begins transcription at the activated adenovirus E4 promoter. The issues addressed include the multiple roles of ATP, the potential effect of polymerase C-terminal domain phosphorylation, and the ability of polymerase to clear the promoter for reinitiation. The results lead to a three-step model for the transition from closed complex to elongation complex, two steps of which use ATP independently. In the first step, studied previously, ATP is hydrolyzed to open the DNA strands over the start site. In a second step, apparently independent of ATP, transcription bubbles move into the initial transcribed region where RNA synthesis can stall. In the third step, transcripts can be made as polymerase is released from these stalled positions with the assistance of an ATP-dependent process, likely phosphorylation of the polymerase C-terminal domain. After this third step, the promoter becomes cleared, allowing for the reinitiation of transcription.

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

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.

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

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.

scholarly journals Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

2013 ◽  
Vol 455 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Michael Aregger ◽  
Victoria H. Cowling
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Translation Initiation ◽  
Transcription Initiation ◽  
Catalytic Domain ◽  
Terminal Domain

The mRNA methyl cap recruits the mediators of processing events and translation initiation. We report that RNMT, the human cap methyltransferase, is recruited to RNA polymerase II via the N-terminal domain and is required for gene expression and cell proliferation.

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

eLife ◽  
2019 ◽  
Vol 8 ◽  
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.

scholarly journals Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations.

Genetics ◽  
1995 ◽  
Vol 140 (4) ◽  
pp. 1223-1233
Author(s):  
M L West ◽  
J L Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Phosphorylation Sites ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Substitution Mutations ◽  
Rna Polymerase Ii Ctd

Abstract The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit plays an essential but poorly understood role in transcription. The CTD is highly phosphorylated in vivo and this modification may be important in the transition from transcription initiation to elongation. We report here the development of a strategy for creating novel yeast CTDs. We have used this approach to show that the minimum viable CTD in yeast contains eight consensus (Tyr1Ser2Pro3Thr4Ser5Pro6Ser7) heptapeptide repeats. Substitution of alanine or glutamate for serines in positions two or five is lethal. In addition, changing tyrosine in position one to phenylalanine is lethal. The effects of mutations that alter potential phosphorylation sites are consistent with a requirement for CTD phosphorylation in vivo.

scholarly journals Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

2021 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Sandra Schilbach ◽  
Shintaro Aibara ◽  
Christian Dienemann ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Complex Structure ◽  
Proximal Part ◽  
Initiation Complex ◽  
Protein Coding ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 Å. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

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