scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

scholarly journals The cryo-electron microscopy structure of the human CDK-activating kinase

2020 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transcription Initiation ◽  
Control Cell ◽  
Regulatory Elements ◽  
Structural Basis ◽  
Control Of Gene Expression ◽  
Cryo Electron Microscopy ◽  
Insight Into ◽  
Cdk Activating Kinase

AbstractThe human CDK-activating kinase (CAK), a complex composed of cyclin dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II subunit Rpb1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell-cycle progression. Here, we have determined the three-dimensional structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and aid in the rational design of therapeutic compounds.SignificanceControl of gene expression and the cell cycle is critical for appropriate cell growth and timely cell division. Failure of the mechanisms regulating these processes can result in proliferative diseases. A molecular complex termed the CDK activating kinase (CAK) impinges on both of these regulatory networks in human cells and is thus a possible drug target for treatment of cancer. Here, we use cryo-electron microscopy to describe the detailed molecular structure of the human CAK, revealing its architecture and the interactions between its regulatory elements. Additionally, we have obtained the structure of the CAK in complex with a small-molecule inhibitor.

scholarly journals Structural basis for CDK7 activation by MAT1 and Cyclin H

2020 ◽  
Author(s):  
Stefan Peissert ◽  
Andreas Schlosser ◽  
Rafaela Kendel ◽  
Jochen Kuper ◽  
Caroline Kisker
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Control Cell ◽  
Model Organism ◽  
Heptad Repeat ◽  
Structural Basis ◽  
Activity Measurements ◽  
Cdk Activating Kinase

AbstractCDK7, Cyclin H, and MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex. CAK is a vital factor for the two essential processes of transcription and cell cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH it phosphorylates the T-loop of CDKs that control cell cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on the MAT1 protein for activation in addition to Cyclin H. We solved the structure of the CAK complex from the model organism C. thermophilum at 2.6 Å resolution. Our structure reveals an intricate network of interactions between MAT1 and its two binding partners CDK7 and Cyclin H providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements combined with functional mutagenesis show that CDK7 activation can occur independently of T-loop phosphorylation and is thus exclusively MAT1 dependent by positioning the CDK7 T-loop in its active conformation. Finally, our structure of the active CAK with a peptide model provides a molecular rationale for heptad repeat phosphorylation.Significance StatementThe fundamental processes of cell cycle regulation and transcription are linked by the heterotrimeric CDK-activating kinase (CAK) complex. We have solved the crystal structure of the active CAK complex and provide a molecular rationale for CAK activation, regulation, and substrate recognition thereby highly advancing our understanding of this essential factor.

scholarly journals Structural basis for CDK7 activation by MAT1 and Cyclin H

2020 ◽  
Vol 117 (43) ◽  
pp. 26739-26748
Author(s):  
Stefan Peissert ◽  
Andreas Schlosser ◽  
Rafaela Kendel ◽  
Jochen Kuper ◽  
Caroline Kisker
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Control Cell ◽  
Model Organism ◽  
Ring Finger ◽  
Structural Basis ◽  
Activity Measurements

Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.

scholarly journals RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

2022 ◽  
Vol 119 (3) ◽  
pp. e2114065119
Author(s):  
Juntaek Oh ◽  
Tiezheng Jia ◽  
Jun Xu ◽  
Jenny Chong ◽  
Peter B. Dervan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Minor Groove ◽  
Dna Lesions ◽  
Structural Basis ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
X Ray ◽  
A Minor ◽  
Insight Into

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

Structural basis of transcription initiation by RNA polymerase II

2015 ◽  
Vol 16 (3) ◽  
pp. 129-143 ◽  
Author(s):  
Sarah Sainsbury ◽  
Carrie Bernecky ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Structural Basis

Structural Biology of RNA Polymerase II Transcription: 20 Years On

2020 ◽  
Vol 36 (1) ◽  
pp. 1-34 ◽  
Author(s):  
Sara Osman ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Focal Point ◽  
Dna Lesions ◽  
Cellular Regulation ◽  
Pol Ii ◽  
Associated Proteins ◽  
Rna Polymerase Ii Transcription

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

scholarly journals Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

2018 ◽  
Vol 115 (11) ◽  
pp. E2538-E2545 ◽  
Author(s):  
Wei Wang ◽  
Celine Walmacq ◽  
Jenny Chong ◽  
Mikhail Kashlev ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Lesions ◽  
Structural Motif ◽  
Structural Basis ◽  
Dependent Manner ◽  
Abasic Site ◽  
Structural Explanation ◽  
Pol Ii ◽  
Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

scholarly journals Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3161-3161
Author(s):  
Vicky Lock ◽  
Laurence Cooke ◽  
Murray Yule ◽  
Neil T Thompson ◽  
K. Della Croce ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Parp Cleavage ◽  
Regulation Of Transcription ◽  
Cytotoxic Effects ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

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