28 research outputs found
Structure of the MED7/MED21 heterodimer and reconstitution of a recombinant Mediator middle module complex
The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. Yeast Mediator has a size of more than one MDa and consists of 25 different polypeptides. Biochemical studies defined three Mediator modules in yeast, the head (MED17) the middle (MED9/MED10) and the tail (MED15) modules. During this work, an E.coli coexpression-copurification system was developed, which allowed to study pairwise interactions of Mediator middle module subunits. With the help of this system I reconstituted a complex of two essential and conserved yeast Mediator middle module proteins, the MED7/MED21 heterodimer, and solved its crystal structure. The heterodimer forms an extended structure, which spans one third of the Mediator length, and almost the diameter of Pol II. It shows a four helix bundle and a coiled-coil protrusion connected by a flexible hinge. Multiple conserved patches can be identified on the surface, which allow for assembly of the middle module. A combination of the coexpression-copurification system and assembly of subcomplexes allowed the reconstitution of a five-subunit Mediator middle module subcomplex. The reconstituted subcomplex is able to bind Pol II in vitro. MED6 associates with the middle module and forms a bridge to the head module. The potential flexibility of this bridge and the MED7/MED21 hinge can account for changes in Mediator structure upon its binding to Pol II or to activators
Halogen Bonds Form the Basis for Selective P-TEFb Inhibition by DRB
SummaryCdk9, the kinase of the positive transcription elongation factor b, is required for processive transcription elongation by RNA polymerase II. Cdk9 inhibition contributes to the anticancer activity of many Cdk inhibitors under clinical investigation and hence there is interest in selective Cdk9 inhibitors. DRB (5,6-dichlorobenzimidazone-1-Ī²-D-ribofuranoside) is a commonly used reagent for Cdk9 inhibition in cell biology studies. The crystal structures of Cdk9 and Cdk2 in complex with DRB reported here describe the molecular basis for the DRB selectivity toward Cdk9. The DRB chlorine atoms form halogen bonds that are specific for the Cdk9 kinase hinge region. Kinetic and thermodynamic experiments validate the structural findings and implicate the C-terminal residues of Cdk9 in contributing to the affinity for DRB. These results open the possibility to exploit halogen atoms in inhibitor design to specifically target Cdk9
The CDK9 C-helix Exhibits Conformational Plasticity That May Explain the Selectivity of CAN508
Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy.In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors.In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.This thesis is not currently available via ORA
Preparation and topology of the Mediator middle module
Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Proteināprotein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes, Med7N/21, Med7C/31 and Med4/9. The subunits, Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4
The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation
Identification, structure, and functional requirement of the Mediator submodule Med7N/31
Mediator is a modular multiprotein complex required for regulated transcription by RNA polymerase (Pol) II. Here, we show that the middle module of the Mediator core contains a submodule of unique structure and function that comprises the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1). The Med7N/31 submodule shows a conserved novel fold, with two proline-rich stretches in Med7N wrapping around the right-handed four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identify specific and overlapping functions of different Mediator submodules
The CDK9 Tail Determines the Reaction Pathway of Positive Transcription Elongation Factor b
The CDK9 C-helix Exhibits Conformational Plasticity That May Explain the Selectivity of CAN508
CDK9 is the kinase of positive transcription elongation
factor
b and facilitates the transition of paused RNA polymerase II to processive
transcription elongation. CDK9 is a validated target for the treatment
of cancer, cardiac hypertrophy, and human immunodeficiency virus.
Here we analyze different CDK9/cyclin T variants to identify a form
of the complex amenable to use in inhibitor design. To demonstrate
the utility of this system, we have determined the crystal structures
of CDK9/cyclin T and CDK2/cyclin A bound to the CDK9-specific inhibitor
CAN508. Comparison of the structures reveals CDK9-specific conformational
changes and identifies a CDK9-specific hydrophobic pocket, adjacent
to the Ī±C-helix. By comparison with a previously published structure
of CDK9/cyclin T/human immunodeficiency virus TAT we find that the
CDK9 Ī±C-helix has a degree of conformational variability that
has the potential to be exploited for inhibitor design