The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Laszlo Tora - One of the best experts on this subject based on the ideXlab platform.
-
Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly
Nature Structural & Molecular Biology, 2018Co-Authors: Simona V. Antonova, Laszlo Tora, Elisabeth Scheer, Matthias Haffke, Eleonora Corradini, Mykolas Mikuciunas, Luca Signor, Kapil Gupta, Robert M. Es, Albert J. R. HeckAbstract:A combination of proteomics and structural analyses reveals the assembly mechanism of transcription factor TFIID in human cells and identifies the chaperonin CCT as a checkpoint in the process. TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5–TAF6–TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6–TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.
-
The architecture of human general transcription factor TFIID core complex
Nature, 2013Co-Authors: Christoph Bieniossek, Laszlo Tora, Patrick Schultz, Gabor Papai, Christiane Schaffitzel, Frederic Garzoni, Maxime Chaillet, Elisabeth Scheer, Petros Papadopoulos, Imre BergerAbstract:The structures of three distinct human transcription factor IID (TFIID) protein assemblies are solved using cryo-electron microscopy; by incorporating TAF8 and TAF10, the key structural changes that remodel TFIID during assembly are determined, particularly the transition from a symmetric core-TFIID to an asymmetric holo-complex. TFIID is the first general transcription factor to bind gene promoters prior to gene transcription by RNA polymerase II, triggering pre-initiation complex formation and functioning as a coactivator. TFIID is a large multiprotein complex composed of TATA-box-binding protein (TBP) and TBP-associated factors (TAFs). Imre Berger and colleagues now determine structures of three distinct human TFIID protein assemblies using cryo-electron microscopy. In their model for step-wise assembly of the complex, the transition from a symmetric core TFIID to an asymmetric holo-complex occurs upon binding of TAF8 and TAF10, which induces major conformational changes. The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks^ 1 , 2 , 3 . TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs)^ 3 . Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID^ 4 , 5 , 6 . A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12)^ 7 . These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy^ 8 , conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8–TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.
-
tfiid taf6 taf9 complex formation involves the heat repeat containing c terminal domain of taf6 and is modulated by TAF5 protein
Journal of Biological Chemistry, 2012Co-Authors: Elisabeth Scheer, Laszlo Tora, Frederic Delbac, Dino Moras, Christophe RomierAbstract:The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.
-
TAF6δ orchestrates an apoptotic transcriptome profile and interacts functionally with p53
BMC Molecular Biology, 2010Co-Authors: Emmanuelle Wilhelm, Laszlo Tora, Mara Kornete, Brice Targat, Jimmy Vigneault-edwards, Mattia Frontini, Arndt Benecke, Brendan BellAbstract:Background TFIID is a multiprotein complex that plays a pivotal role in the regulation of RNA polymerase II (Pol II) transcription owing to its core promoter recognition and co-activator functions. TAF6 is a core TFIID subunit whose splice variants include the major TAF6α isoform that is ubiquitously expressed, and the inducible TAF6δ. In contrast to TAF6α, TAF6δ is a pro-apoptotic isoform with a 10 amino acid deletion in its histone fold domain that abolishes its interaction with TAF9. TAF6δ expression can dictate life versus death decisions of human cells. Results Here we define the impact of endogenous TAF6δ expression on the global transcriptome landscape. TAF6δ was found to orchestrate a transcription profile that included statistically significant enrichment of genes of apoptotic function. Interestingly, gene expression patterns controlled by TAF6δ share similarities with, but are not equivalent to, those reported to change following TAF9 and/or TAF9b depletion. Finally, because TAF6δ regulates certain p53 target genes, we tested and demonstrated a physical and functional interaction between TAF6δ and p53. Conclusion Together our data define a TAF6δ-driven apoptotic gene expression program and show crosstalk between the p53 and TAF6δ pathways.
-
Mapping key functional sites within yeast TFIID
The EMBO Journal, 2004Co-Authors: Claire Leurent, Steven L. Sanders, Màté A. Demény, Krassimira A. Garbett, Christine Ruhlmann, P. Anthony Weil, Laszlo Tora, Patrick SchultzAbstract:The transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a key role in the regulation of gene expression by RNA polymerase II. The structure of yeast TFIID, as determined by electron microscopy and digital image analysis, is formed by three lobes, labelled A–C, connected by thin linking domains. Immunomapping revealed that TFIID contains two copies of the WD-40 repeat-containing TAF5 and that TAF5 contributes to the linkers since its C- and N-termini were found in different lobes. This property was confirmed by the finding that a recombinant complex containing TAF5 complexed with six histone fold containing TAFs was able to form a trilobed structure. Moreover, the N-terminal domain of TAF1 was mapped in lobe C, whereas the histone acetyltransferase domain resides in lobe A along with TAF7. TBP was found in the linker domain between lobes A and C in a way that the N-terminal 100 residues of TAF1 are spanned over it. The implications of these data with regard to TFIID function are discussed.
Elisabeth Scheer - One of the best experts on this subject based on the ideXlab platform.
-
Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly.
Nature Structural & Molecular Biology, 2018Co-Authors: Simona V. Antonova, Matthias Haffke, Eleonora Corradini, Mykolas Mikuciunas, Luca Signor, Kapil Gupta, Elisabeth ScheerAbstract:TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.
-
Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly
Nature Structural & Molecular Biology, 2018Co-Authors: Simona V. Antonova, Laszlo Tora, Elisabeth Scheer, Matthias Haffke, Eleonora Corradini, Mykolas Mikuciunas, Luca Signor, Kapil Gupta, Robert M. Es, Albert J. R. HeckAbstract:A combination of proteomics and structural analyses reveals the assembly mechanism of transcription factor TFIID in human cells and identifies the chaperonin CCT as a checkpoint in the process. TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5–TAF6–TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6–TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.
-
Cytoplasmic TAF2–TAF8–TAF10 complex provides evidence for nuclear holo–TFIID assembly from preformed submodules
Nature Communications, 2015Co-Authors: Simon Trowitzsch, Gabor Papai, Christiane Schaffitzel, Elisabeth Scheer, Cristina Viola, Sascha Conic, Virginie Chavant, Marjorie Fournier, Ima-obong Ebong, Matthias HaffkeAbstract:General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID—a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)—assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8–TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core–TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo–TFIID, regulated by nuclear import of preformed cytoplasmic submodules. TFIID is an essential transcription factor complex that controls the expression of most protein-coding genes in eukaryotes. Here the authors identify and characterize a complex containing TAF2, TAF8 and TAF10, which assembles in the cytoplasm before integration into the nuclear holo–TFIID complex.
-
The architecture of human general transcription factor TFIID core complex
Nature, 2013Co-Authors: Christoph Bieniossek, Laszlo Tora, Patrick Schultz, Gabor Papai, Christiane Schaffitzel, Frederic Garzoni, Maxime Chaillet, Elisabeth Scheer, Petros Papadopoulos, Imre BergerAbstract:The structures of three distinct human transcription factor IID (TFIID) protein assemblies are solved using cryo-electron microscopy; by incorporating TAF8 and TAF10, the key structural changes that remodel TFIID during assembly are determined, particularly the transition from a symmetric core-TFIID to an asymmetric holo-complex. TFIID is the first general transcription factor to bind gene promoters prior to gene transcription by RNA polymerase II, triggering pre-initiation complex formation and functioning as a coactivator. TFIID is a large multiprotein complex composed of TATA-box-binding protein (TBP) and TBP-associated factors (TAFs). Imre Berger and colleagues now determine structures of three distinct human TFIID protein assemblies using cryo-electron microscopy. In their model for step-wise assembly of the complex, the transition from a symmetric core TFIID to an asymmetric holo-complex occurs upon binding of TAF8 and TAF10, which induces major conformational changes. The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks^ 1 , 2 , 3 . TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs)^ 3 . Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID^ 4 , 5 , 6 . A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12)^ 7 . These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy^ 8 , conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8–TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.
-
tfiid taf6 taf9 complex formation involves the heat repeat containing c terminal domain of taf6 and is modulated by TAF5 protein
Journal of Biological Chemistry, 2012Co-Authors: Elisabeth Scheer, Laszlo Tora, Frederic Delbac, Dino Moras, Christophe RomierAbstract:The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.
F R Mcfeely - One of the best experts on this subject based on the ideXlab platform.
-
x ray photoelectron spectroscopic studies of chemical vapor deposition with TAF5 precursor on polyimide surfaces
Journal of Applied Physics, 1992Co-Authors: D Ugolini, S P Kowalczyk, F R McfeelyAbstract:Chemical vapor deposition of Ta on the polyimide pyromellitic dianhydride oxydianiline was attempted using TAF5 as the precursor. The deposition temperature was 350 °C. Photoelectron spectroscopy measurements show that Ta is bound in a high oxidation state. Our data show strong evidence that initially the ternary compound TaOF3 is formed on polyimide in a first step. The carbonyl group is the main reactive site upon TAF5 exposure. It has to be noted that no passivation of the surface takes place and that there is no limit for film thickness. At very high doses, however, the film concentration at the surface is suggestive for the stable compound TaO2F. This observation is explained in terms of the formation of a metastable TaOF3 compound in the first stages of film growth and a phase transformation to TaO2F at larger thicknesses. Annealing experiments to 500 °C suggest the transformation to the oxide Ta2O5 that is strongly related to the almost complete loss of fluorine from the film upon heating.
-
X‐ray photoelectron spectroscopic studies of chemical vapor deposition with TAF5 precursor on polyimide surfaces
Journal of Applied Physics, 1992Co-Authors: D Ugolini, S P Kowalczyk, F R McfeelyAbstract:Chemical vapor deposition of Ta on the polyimide pyromellitic dianhydride oxydianiline was attempted using TAF5 as the precursor. The deposition temperature was 350 °C. Photoelectron spectroscopy measurements show that Ta is bound in a high oxidation state. Our data show strong evidence that initially the ternary compound TaOF3 is formed on polyimide in a first step. The carbonyl group is the main reactive site upon TAF5 exposure. It has to be noted that no passivation of the surface takes place and that there is no limit for film thickness. At very high doses, however, the film concentration at the surface is suggestive for the stable compound TaO2F. This observation is explained in terms of the formation of a metastable TaOF3 compound in the first stages of film growth and a phase transformation to TaO2F at larger thicknesses. Annealing experiments to 500 °C suggest the transformation to the oxide Ta2O5 that is strongly related to the almost complete loss of fluorine from the film upon heating.
-
photoelectron spectroscopy studies of chemical vapor deposition of ta from a TAF5 precursor on si and sio2 substrates
Journal of Applied Physics, 1991Co-Authors: D Ugolini, S P Kowalczyk, F R McfeelyAbstract:The increasing need for metallization of microelectronic structures involving deep trenches and high aspect ratio features has sparked considerable interest in chemical vapor deposition metallization schemes. In this work the results of photoemission investigations of the substrate‐driven reduction of TAF5 to Ta metal on Si(111) and SiO2 substrates are reported. At moderate temperatures the reaction showed no selectivity between these two substrates, in contrast to the process with the similar molecule WF6. At 400 °C metallic Ta films could be grown on both Si and SiO2; however, x‐ray photoelectron spectroscopy measurements show that the bonding of the film to Si(111) is dominated by fluorine atoms at the interface, whereas film growth on SiO2 tends to form an oxide interface with TaOF3 stoichiometry. Annealing of the deposited film to about 700 °C leads to Si diffusion to the surface which is accompanied by the release of fluorine from the film. The deposition of TAF5 on Si(111) at 250 °C results initial...
-
comparison of the selective adsorption and reactivity behavior of wf6 and TAF5 on sio2 and polyimide surfaces
Applied Physics Letters, 1990Co-Authors: F R Mcfeely, L J Terminello, S P KowalczykAbstract:The initial stages of WF6 and TAF5 adsorption on SiO2 and polyimide surfaces were investigated by photoelectron spectroscopy. WF6 selectively adsorbs on Si relative to SiO2 and polyimide, while TAF5 exhibited nonselective adsorption behavior. This trend is explained by differences in molecular structure and suggests a general basis for predicting selective deposition.
Frank Endres - One of the best experts on this subject based on the ideXlab platform.
-
The Au(111)/IL interfacial nanostructure in the presence of precursors and its influence on the electrodeposition process.
Faraday Discussions, 2017Co-Authors: Natalia Borisenko, Abhishek Lahiri, Giridhar Pulletikurthi, Timo Carstens, Janine Zahlbach, Rob Atkin, Frank EndresAbstract:Ionic liquids have attracted significant interest as electrolytes for the electrodeposition of metals and semiconductors, but the details of the deposition processes are not yet well understood. In this paper, we give an overview of how the addition of various precursors (TAF5, SiCl4, and GaCl3) affects the solid/IL interfacial structure. In situ Atomic Force Microscopy (AFM) and vibrational spectroscopy have been employed to study the changes of the Au(111)/IL interface and in the electrolytes, respectively. Ionic liquids with the 1-butyl-1-methylpyrrolidinium ([Py1,4]+) cation and bis(trifluoromethylsulfonyl)amide ([TFSA]−), trifluoromethylsulfonate ([TfO]−) and tris(pentafluoroethyl)trifluorophosphate ([FAP]−) as anions were chosen for this purpose. In situ AFM force–distance measurements reveal that both the anion of the IL and the solutes (TAF5 or GaCl3) influence the Electrical Double Layer (EDL) structure of the Au(111)/IL interface, which can affect the deposition process of Ta and the morphology of the Ga electrodeposits, respectively. Furthermore, the concentration of the precursor can significantly alter the Au(111)/[Py1,4][FAP]–SiCl4 interfacial structure wherein the presence of 0.25 M SiCl4 a double layer structure forms that facilitates Si deposition. This study may provide some critical insights into the structure of the electrode/IL interface for specific applications.
-
Electroreduction of tantalum fluoride in a room temperature ionic liquid at variable temperatures.
Physical Chemistry Chemical Physics, 2005Co-Authors: S. Zein El Abedin, H. K. Farag, E. M. Moustafa, Urs Welz-biermann, Frank EndresAbstract:The present paper deals with the electroreduction of TAF5 in the room temperature ionic liquid 1-butyl-1-methyl-pyrrolidinium bis(tri-fluoromethylsulfonyl)imide ([BMP]Tf2N) at different temperatures for the sake of electrodeposition of tantalum. The study was carried out using cyclic voltammetry and chronoamperometry measurements complemented by SEM-EDAX and XRD investigations. In situ scanning tunneling microscopy and I–U tunneling spectroscopy were also utilized for characterization of the electrodeposits. The results show that, in addition to the formation of insoluble compounds, Ta can be electrodeposited in the ionic liquid ([BMP]Tf2N) containing 0.5 M TAF5 at 200 °C on polycrystalline Pt and Au(111) electrodes. By addition of LiF to the electrolyte, the quality and the adherence of the electrodeposit were found to be improved. An in situI–U tunneling spectrum with about 300 nm thickness of the electrodeposit shows metallic behaviour indicating the formation of elemental tantalum. Moreover, the XRD patterns of the electrodeposit, obtained potentiostatically at −1.8 V (vs. Pt) in ([BMP]Tf2N) containing 0.25 M TAF5 and 0.25 M LiF on Pt electrode at 200 °C, show the characteristic patterns of crystalline tantalum.
-
Electroreduction of tantalum fluoride in a room temperature ionic liquid at variable temperatures
Physical Chemistry Chemical Physics, 2005Co-Authors: S. Zein El Abedin, H. K. Farag, E. M. Moustafa, Urs Welz-biermann, Frank EndresAbstract:The present paper deals with the electroreduction of TAF5 in the room temperature ionic liquid 1-buty-1-methyl-pyrrolidinium bis(tri-fluoromethylsulfonyl)imide ([BMP]Tf2N) at different temperatures for the sake of electrodeposition of tantalum. The study was carried out using cyclic voltammetry and chronoamperometry measurements complemented by SEM-EDAX and XRD investigations. In situ scanning tunneling microscopy and I-U tunneling spectroscopy were also utilized for characterization of the electrodeposits. The results show that, in addition to the formation of insoluble compounds, Ta can be electrodeposited in the ionic liquid ([BMP]Tf2N) containing 0.5 M TAF5 at 200 degrees C on polycrystalline Pt and Au(111) electrodes. By addition of LiF to the electrolyte, the quality and the adherence of the electrodeposit were found to be improved. An in situ I-U tunneling spectrum with about 300 nm thickness of the electrodeposit shows metallic behaviour indicating the formation of elemental tantalum. Moreover, the XRD patterns of the electrodeposit, obtained potentiostatically at -1.8 V (vs. Pt) in ([BMP]Tf2N) containing 0.25 M TAF5 and 0.25 M LiF on Pt electrode at 200 degrees C, show the characteristic patterns of crystalline tantalum.
Matthias Haffke - One of the best experts on this subject based on the ideXlab platform.
-
Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly.
Nature Structural & Molecular Biology, 2018Co-Authors: Simona V. Antonova, Matthias Haffke, Eleonora Corradini, Mykolas Mikuciunas, Luca Signor, Kapil Gupta, Elisabeth ScheerAbstract:TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5-TAF6-TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6-TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.
-
Chaperonin CCT checkpoint function in basal transcription factor TFIID assembly
Nature Structural & Molecular Biology, 2018Co-Authors: Simona V. Antonova, Laszlo Tora, Elisabeth Scheer, Matthias Haffke, Eleonora Corradini, Mykolas Mikuciunas, Luca Signor, Kapil Gupta, Robert M. Es, Albert J. R. HeckAbstract:A combination of proteomics and structural analyses reveals the assembly mechanism of transcription factor TFIID in human cells and identifies the chaperonin CCT as a checkpoint in the process. TFIID is a cornerstone of eukaryotic gene regulation. Distinct TFIID complexes with unique subunit compositions exist and several TFIID subunits are shared with other complexes, thereby conveying precise cellular control of subunit allocation and functional assembly of this essential transcription factor. However, the molecular mechanisms that underlie the regulation of TFIID remain poorly understood. Here we use quantitative proteomics to examine TFIID submodules and assembly mechanisms in human cells. Structural and mutational analysis of the cytoplasmic TAF5–TAF6–TAF9 submodule identified novel interactions that are crucial for TFIID integrity and for allocation of TAF9 to TFIID or the Spt-Ada-Gcn5 acetyltransferase (SAGA) co-activator complex. We discover a key checkpoint function for the chaperonin CCT, which specifically associates with nascent TAF5 for subsequent handover to TAF6–TAF9 and ultimate holo-TFIID formation. Our findings illustrate at the molecular level how multisubunit complexes are generated within the cell via mechanisms that involve checkpoint decisions facilitated by a chaperone.
-
Cytoplasmic TAF2–TAF8–TAF10 complex provides evidence for nuclear holo–TFIID assembly from preformed submodules
Nature Communications, 2015Co-Authors: Simon Trowitzsch, Gabor Papai, Christiane Schaffitzel, Elisabeth Scheer, Cristina Viola, Sascha Conic, Virginie Chavant, Marjorie Fournier, Ima-obong Ebong, Matthias HaffkeAbstract:General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID—a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)—assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8–TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core–TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo–TFIID, regulated by nuclear import of preformed cytoplasmic submodules. TFIID is an essential transcription factor complex that controls the expression of most protein-coding genes in eukaryotes. Here the authors identify and characterize a complex containing TAF2, TAF8 and TAF10, which assembles in the cytoplasm before integration into the nuclear holo–TFIID complex.
-
Crystal structure of human TAF5/TAF6/TAF9 complex at 2.7 A resolution
Acta Crystallographica Section A, 2014Co-Authors: Matthias Haffke, Imre BergerAbstract:Human general transcription factor TFIID is a megadalton sized multiprotein complex containing the TATA-box binding protein (TBP) and 13 TBP Associated Factors (TAFs). Structural studies by cryo-electron microscopy (cryo-EM) have uncovered the overall architecture of TFIID, providing valuable insight into subunit assembly and promoter recognition at medium resolution. Despite considerable effort, high resolution structural information of TAFs to date have been largely limited to structures of individual domains and assemblies of TAF histone-fold pairs, which are prevalent in TFIID. We used our MultiBac baculovirus/insect cell technology to obtain recombinant TFIID complexes including holo-TFIID in unprecedented quality and quantity, setting the stage for high resolution structural analysis by X-ray crystallography. We identified stable subassemblies of the TFIID complexes by limited proteolysis, and utilized a rapid combinatorial co-expression approach to produce and purify specimens for high-throughput crystallization. We crystallized a thus defined complex formed by TAF5, TAF6 and TAF9 and determined the structure to a resolution of 2.7 Å, revealing intricate molecular interactions at the core of human TFIID.
