Periodic Reporting for period 1 - POL2-TFIID (The impact of TFIID on the transcription initiation machinery – a structural perspective.)
Okres sprawozdawczy: 2016-03-01 do 2018-02-28
Transcription is a first step of gene expression, a process by which a functional gene product is synthetized based on genetic information from DNA. Gene expression is tightly regulated with complex mechanisms which give cells the flexibility to adapt to a variable environment. Defects in regulation and transcription mechanisms lead to serious pathologies in cells and organisms, which in humans include cancer development, autoimmunity and neurological disorders, cardiovascular diseases etc. Last two decades brought a great development in the understanding mechanisms involved in transcription, especially the first step of the process - transcription initiation. It is also a stage which is greatly involved in the gene regulation process.
Transcription of protein-coding genes in eukaryotic organisms starts with stepwise formation of the pre-initiation complex (PIC) comprising RNA polymerase II (Pol II) and the general transcription factors (GTFs) on promoter DNA. According to a classical model, during the first step of PIC assembly the TATA-box binding protein (TBP), a subunit of the TFIID complex, recognizes promoter DNA. The interaction is supported by TFIIB, which is also required for recruiting the Pol II – TFIIF complex to the promoter. PIC formation is completed with binding of transcription factors TFIIE and TFIIH. Particular elements of the transcription initiation complexes as well as the complete machinery itself were subjects of structural studies for many years resulting in a priceless insight into transcription initiation mechanisms. The resent great achievements include the structure of PIC (consisting of promoter DNA scaffold, Pol II, TFIIA, -B, -E, -F, -H, TBP) together with a co-factor Mediator. However, the very first step of the transcription initiation, namely the recognition of the promoter DNA and initialisation of the PIC assembly by TFIID, remains not fully explored. It is crucial to learn more about this GTF because it also serves as a co-factor combining signals from distal-acting activators and repressors with the recognition of core promoter sequences, thus plays important role in gene regulation.
Although TFIID has been studied for a long time, only a moderate resolution structures are available. It is a large, multi-subunit complex composed of TBP and 13-14 TBP-associated factors (TAFs) reaching approximately 1.2 MDa. TFIID is very flexible, dynamic, and its subunits contain a lot of intrinsically disordered regions which makes it a challenging target for structural studies. This project focused on determination of the architecture of TFIID in complex with Pol II, promoter DNA and additional general transcription factors (TFIIA, TFIIB and TFIIF) followed by biochemical, and functional studies of its impact on the other components of transcription initiation machinery. Such information is necessary to understand the role of TFIID in the transcription initiation and gene regulation.
As an outcome of the project, a protocol for the endogenous purification of TFIID was established yielding the material of high purity and homogeneity. We have also established a yeast over-expression system capable of co-expressing all TFIID subunits to obtain more material for structural studies. The promising candidates for promoter scaffolds (both containing TATA box and TATA-less) were selected using bioinformatical analysis. Moreover, we managed to assemble TFIID together with Pol II and TFIIA, -B, -F, -E on the DNA scaffold using several alternative approaches. The comprehensive structural analysis of the holo-complex is in progress and will provide better understanding of not only transcription initiation mechanism but also the cross-talk between gene regulation and transcription elements.
Transcription of protein-coding genes in eukaryotic organisms starts with stepwise formation of the pre-initiation complex (PIC) comprising RNA polymerase II (Pol II) and the general transcription factors (GTFs) on promoter DNA. According to a classical model, during the first step of PIC assembly the TATA-box binding protein (TBP), a subunit of the TFIID complex, recognizes promoter DNA. The interaction is supported by TFIIB, which is also required for recruiting the Pol II – TFIIF complex to the promoter. PIC formation is completed with binding of transcription factors TFIIE and TFIIH. Particular elements of the transcription initiation complexes as well as the complete machinery itself were subjects of structural studies for many years resulting in a priceless insight into transcription initiation mechanisms. The resent great achievements include the structure of PIC (consisting of promoter DNA scaffold, Pol II, TFIIA, -B, -E, -F, -H, TBP) together with a co-factor Mediator. However, the very first step of the transcription initiation, namely the recognition of the promoter DNA and initialisation of the PIC assembly by TFIID, remains not fully explored. It is crucial to learn more about this GTF because it also serves as a co-factor combining signals from distal-acting activators and repressors with the recognition of core promoter sequences, thus plays important role in gene regulation.
Although TFIID has been studied for a long time, only a moderate resolution structures are available. It is a large, multi-subunit complex composed of TBP and 13-14 TBP-associated factors (TAFs) reaching approximately 1.2 MDa. TFIID is very flexible, dynamic, and its subunits contain a lot of intrinsically disordered regions which makes it a challenging target for structural studies. This project focused on determination of the architecture of TFIID in complex with Pol II, promoter DNA and additional general transcription factors (TFIIA, TFIIB and TFIIF) followed by biochemical, and functional studies of its impact on the other components of transcription initiation machinery. Such information is necessary to understand the role of TFIID in the transcription initiation and gene regulation.
As an outcome of the project, a protocol for the endogenous purification of TFIID was established yielding the material of high purity and homogeneity. We have also established a yeast over-expression system capable of co-expressing all TFIID subunits to obtain more material for structural studies. The promising candidates for promoter scaffolds (both containing TATA box and TATA-less) were selected using bioinformatical analysis. Moreover, we managed to assemble TFIID together with Pol II and TFIIA, -B, -F, -E on the DNA scaffold using several alternative approaches. The comprehensive structural analysis of the holo-complex is in progress and will provide better understanding of not only transcription initiation mechanism but also the cross-talk between gene regulation and transcription elements.
We screened numerous amount of tagging systems to identify the optimal approach to purify TFIID from the endogenous source (S. cerevisiae). Obtaining a high-quality sample enabled the assembly of the holo-complex including promoter scaffold, Pol II, TFIIA, -B, -D, -E, -F. We established several protocols for the assembly to improve the homogeneity and the concentration of the final holo-complex. A successful assembling of the holo-complex allowed us to attempt initial sample analysis with transmission electron microscopy (TEM). Furthermore, the yeast over-expression system for TFIID was developed to improve the yield of the complex as well as the expression of sub-complexes. Several stable sub-complexes have been determined and they are subjects of crystallographic studies.
Several publications disseminating the results are expected to be published in the future. The results of the project were presented or discussed at conferences such as Keystone Symposia conference Cryo-EM from Cells to Molecules: Multi-Scale Visualization of Biological Systems 2018; Prato cryoEM course 2018 and Marie Curie Alumni Association General Assembly and Conference 2017.
The project enabled the development of the Fellow towards becoming independent researcher by providing opportunities to broaden methods range (biophysical methods, structural data processing) and improve leadership and project management abilities thanks to a possibility to supervise students and a technician.
Several publications disseminating the results are expected to be published in the future. The results of the project were presented or discussed at conferences such as Keystone Symposia conference Cryo-EM from Cells to Molecules: Multi-Scale Visualization of Biological Systems 2018; Prato cryoEM course 2018 and Marie Curie Alumni Association General Assembly and Conference 2017.
The project enabled the development of the Fellow towards becoming independent researcher by providing opportunities to broaden methods range (biophysical methods, structural data processing) and improve leadership and project management abilities thanks to a possibility to supervise students and a technician.
For the first time all S.cerevisiae genes of TFIID are co-expressed in the inducible yeast over-expression system. It greatly improves the yield of the sample and thus facilitates biophysical characterization of the complex and its interactions with other elements of PIC. It also makes any modifications of subunits including truncations, mutations and tagging very easy. The developed over-expression system is suitable for expression of other proteins and even large protein complexes. It is used to obtain protein material for other projects in the department. The project was used to train the Fellow, other research staff and students. Furthermore, by participating in the Marie Curie Alumni Association within Polish and German Chapters the Fellow has positively influenced development of the European scientific society and could impact actions taken to improve situation of European scientists as well as acquired tools important in popularization of the science within the European Research Area.