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.
