Zooming in on Transcription Preinitiation

AbstractClass II gene transcription commences with the assembly of the Preinitiation Complex (PIC) from a plethora of proteins and protein assemblies in the nucleus, including the General Transcription Factors (GTFs), RNA polymerase II (RNA pol II), co-activators, co-repressors, and more. TFIID, a megadalton-sized multiprotein complex comprising 20 subunits, is among the first GTFs to bind the core promoter. TFIID assists in nucleating PIC formation, completed by binding of further factors in a highly regulated stepwise fashion. Recent results indicate that TFIID itself is built from distinct preformed submodules, which reside in the nucleus but also in the cytosol of cells. Here, we highlight recent insights in transcription factor assembly and the regulation of transcription preinitiation

Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly

International audienc

Etudes structurales du facteur de transcription général humain TFIID CORE-COMPLEXES

La transcription des gènes humains par la polymérase II (Pol II) commence avec l'assemblage du complexe de pré-initiation (PIC) au niveau du promoteur. L'assemblage du PIC est initié par le facteur de transcription général TFIID, un complexe de protéines faisant 1 mégadalton et contenant TBP et 13 facteurs TBP-associés (TAF). Des études structurales sur TFIID par cryo-EM ont révélé l'architecture globale de TFIID, donnant un aperçu de l'assemblage des sous-unités et la reconnaissance du promoteur à faible et moyenne résolution. Cependant, les structures à haute résolution de plusieurs TAF ne sont pas disponibles à ce jour, ce qui limite notre compréhension actuelle des interactions TAF-TAF et du mécanisme d'assemblage de TFIID.J'ai utilisé une stratégie de co-expression dans des cellules d'insectes sur la base de notre système MultiBac afin d'obtenir des sous-complexes recombinants TFIID de qualité sans précédent et de quantité suffisante pour des études structurales. J'ai cristallisé un complexe contenant les TAFs TAF5, TAF6 et TAF9 et déterminé sa structure à une résolution de 2.7 Å. Notre structure donne des aperçus détaillés sur les interactions de ces sous-unités essentiels de TFIID: La structure révèle des interactions serrés inattendues entre le domaine N-terminal et le domaine à répétition WD40 contenant 7 lames de TAF5 avec le domaines "histone fold" (HFDs) de TAF6/9. Les interactions entre ces domaines conservés inter-espèces expliquent en détail comment TAF5 sert de plate-forme de liaison pour les HFDs dans TFIID. Cette étude étend notre compréhension de l'échafaudage moléculaire au noyau central de la version humaine de TFIID, ce qui est essentiel pour la formation de holo-TFIID.De plus, j'ai pu isoler un domaine structural conservé de la version humaine de TAF6, le domaine à répétition HEAT de TAF6 en utilisant une approche de protéolyse limitée sur des core-complexes TAF5/6/9 prédéfinis. J'ai pu déterminer la structure du domaine à répétition HEAT de TAF6 jusqu'à 2,0 Å de résolution. La structure offre un éclairage supplémentaire sur l'architecture de la version humaine de TFIID et révèle la présence d'une grande zone chargée positivement sur sa surface, probablement impliqué dans la liaison à l'ADN du noyau promoteur liaison.Basé sur les structures résolues et les informations obtenues dans des expériences de protéolyse limitée, j'ai pu identifier les complexes minimaux de la base du complexe TFIID humain (TAF 4/5/6/9/12). J'ai identifié les HFDs de l'hétérodimère TAF4/12 comme étant essentielle à la formation d'un complexe avec TAF5/6/9. Ces résultats guideront la conception future de construction pour la cristallisation du complexe humain TFIID de base, améliorant ainsi nos connaissances sur le mécanisme d'assemblage de TFIID ainsi que sur l'architecture de ses principaux composants à une résolution atomique.Human gene transcription by Polymerase II (PolII) begins with the assembly of the pre-initiation complex (PIC) at the promoter. PIC assembly is initiated by the general transcription factor TFIID, a megadalton sized protein complex containing TBP and 13 TBP associated Factors (TAFs).\u2028Structural studies on TFIID by cryo-EM have revealed the overall architecture of TFIID, providing insights into subunit assembly and promoter recognition at low to medium resolution. However, high-resolution structures of several TAFs are not available to date, limiting our current understanding of TAF-TAF interactions and the assembly mechanism of TFIID.I used a co-expression strategy in insect cells based on our MultiBac system to obtain recombinant TFIID sub-complexes of unprecedented quality and quantity for structural studies. I crystallized a complex containing the TAFs TAF5, TAF6 and TAF9 and determined its structure up to a resolution of 2.7 Å. Our structure gives detailed insights into the interactions of these essential TFIID subunits: The structure reveals unexpected tight interactions between the N-terminal domain and the 7-bladed WD40 repeat domain of TAF5 with the TAF6/9 histone fold domains (HFDs). The interactions between these inter-species conserved domains explain in detail how TAF5 serves as a binding platform for HFDs in TFIID. This study extends our understanding of the molecular scaffold at the central core of human TFIID, which is essential for holo-TFIID formation.Additionally, I could isolate a conserved structural domain of human TAF6, the TAF6 HEAT repeat domain by using a limited proteolysis approach on predefined TAF5/6/9 core-complexes. I could determine the structure of the TAF6 HEAT repeat domain up to 2.0 Å resolution. The structure provides additional insights into the architecture of human TFIID and reveals the presence of a large positively charged patch on its surface, probably involved in core-promoter DNA binding.Based on the obtained structures and information gained in limited proteolysis experiments, I was able to identify minimal complexes of human core-TFIID (TAFs 4/5/6/9/12). I identified the HFDs of the TAF4/12 heterodimer to be essential for the complex formation with TAF5/6/9. These findings will guide future construct design for crystallization of human core-TFIID, enhancing our knowledge about the TFIID assembly mechanism and the architecture of its core-components at atomic resolution

Experimental Investigation of Concrete Sandwich Walls with Glass-Fiber-Composite Connectors Exposed to Fire and Mechanical Loading

Precast concrete sandwich panels (PCSPs) are known for their good thermal, acoustic and structural properties. Severe environmental demands can be met by PCSPs due to their use of highly thermally insulating materials and non-metallic connectors. One of the main issues limiting the wider use of sandwich walls in construction is their unknown fire resistance. Furthermore, the actual behaviour of connectors and insulation in fire in terms of their mechanical performance and their impact on fire spread and the fire resistance of walls is not fully understood. This paper presents an experimental investigation on the structural and thermal behaviour of PCSPs with mineral-wool insulation and glass-fiber-reinforced polymeric bar connectors coupling two concrete wythes. Three full-size walls were tested following the REI certification test procedure for fire walls under fire and vertical eccentric and post-fire mechanical impact load. The three test configurations were adopted for the assessment of the connectors’ fire behaviour and its impact on the general fire resistance of the walls. All the specimens met the REI 120-M criteria. The connectors did not contribute to the fire’s spread and the integrity of the walls was maintained throughout the testing time. This was also confirmed in the most unfavourable test configuration, in which some of the connectors in the inner area of the wall were significantly damaged, and yet the structural connection of the concrete wythes was maintained. The walls experienced heavy heat-induced thermal bowing. The significant contribution of connectors to the stiffness of the wall during fire was observed and discussed

Experimental investigation of the static and fatigue bond behaviour of GFRP bars and concrete

The experimental investigation deals with the quasi-static and fatigue behavior of the bond between GFRP (glass fibre reinforced polymer) rebars and concrete. Eccentric pull-out test set-up was adopted to measure the effect of concrete cover and concrete mechanical properties. For cycling tests, the ratio R of the maximum load in the cycle to the static strength was also varied to estimate the fatigue re-sistance under different load levels. GFRP rebars were considered of diameter 8 mm. Specimens were cast with two different concrete classes and bars were positioned according to two concrete covers. Cyclic loading was performed with two load levels. Quasi-static results show similar shear strength for the different concrete covers and the shear strength increases with the concrete mechanical proper-ties. Cyclic tests provide a fatigue life exceeding one million cycles for the minimum load level con-sidered while for the maximum the number of cycles to failure extremely increase considering the higher concrete quality

Experimental Investigation of Concrete Sandwich Walls with Glass-Fiber-Composite Connectors Exposed to Fire and Mechanical Loading

Precast concrete sandwich panels (PCSPs) are known for their good thermal, acoustic and structural properties. Severe environmental demands can be met by PCSPs due to their use of highly thermally insulating materials and non-metallic connectors. One of the main issues limiting the wider use of sandwich walls in construction is their unknown fire resistance. Furthermore, the actual behaviour of connectors and insulation in fire in terms of their mechanical performance and their impact on fire spread and the fire resistance of walls is not fully understood. This paper presents an experimental investigation on the structural and thermal behaviour of PCSPs with mineral-wool insulation and glass-fiber-reinforced polymeric bar connectors coupling two concrete wythes. Three full-size walls were tested following the REI certification test procedure for fire walls under fire and vertical eccentric and post-fire mechanical impact load. The three test configurations were adopted for the assessment of the connectors’ fire behaviour and its impact on the general fire resistance of the walls. All the specimens met the REI 120-M criteria. The connectors did not contribute to the fire’s spread and the integrity of the walls was maintained throughout the testing time. This was also confirmed in the most unfavourable test configuration, in which some of the connectors in the inner area of the wall were significantly damaged, and yet the structural connection of the concrete wythes was maintained. The walls experienced heavy heat-induced thermal bowing. The significant contribution of connectors to the stiffness of the wall during fire was observed and discussed

Static and fatigue bond behaviour of GFRP bars and concrete

The experimental investigation is intended to study the influence of some parameters on the quasi-static and the fatigue behaviour of the bond between glass fibre reinforced plastic (GFRP) rebars and concrete. This is an important aspect in FRP reinforced concrete structural elements and of relevant importance in thin reinforced concrete panels extensively adopted as façade or pavements. The pull-out set-up with eccentrically positioned GFRP ComBAR® bar was adopted to measure the effect of two parameters on the bond mechanical features: thickness of the concrete cover and concrete mechanical properties. For cycling tests, another parameter, the maximum load in the cycle, was introduced to estimate the fatigue life under different load levels. Quasi-static tests showed that concrete compressive strength influence on bond properties is much more pronounced than that of the concrete cover. Cyclic tests at highest load level revealed that higher concrete quality attained longer fatigue life

Concrete cover effect on the bond of GFRP bar and concrete under static loading

Paper presents assessment of bond behaviour between GFRP bars and concrete, investigated through set of centric and eccentric pull-out specimens. Main parameters under investigation are 1) bar external surface, 2) concrete mechanical properties and 3) concrete cover. Corresponding tests with steel reinforcement are performed for comparison in some cases. DIC technique was used for recording and evaluating of strain field on frontal side of eccentric specimens. Consequently, cracking patterns and local bond behaviour are described in details. Increasing of concrete mechanical properties always enhanced bond strength and delayed cracking of concrete cover. Ribbed GFRP bars showed excellent bonding performance when combined with low concrete cover. Their low splitting tendency and specific rib geometry developed better bond behaviour in case of eccentric tests, which showed the possibility of a proper prediction of the bonding behaviour of structural components