Initiation of RNA polymerase II (Pol II) transcription is activated by the assembly of preinitiation complex (PIC) onto gene promoters. PIC contains the largest general transcription factor (GTF), TFIID that comprises TATA box-binding protein (TBP). The binding of TBP to TATA promoter is rate-limiting step in PIC assembly and is stabilized by GTFs, TFIIA and TFIIB. TFIID also contains reader domains that recognize specific epigenetic marks in nucleosomes located upstream and downstream of transcription start site. Structural information on the architecture of nucleosomes, DNA, TFIIA, TFIIB and TFIID in this context remains elusive to date limiting our understanding of DNA architecture at promoter site and the interaction of TFIID with epigenetically modified nucleosomes in transcription initiation. The objective of this proposal is to explore this cross-talk by creating a synthetic DNA super-template for PIC assembly containing modified nucleosomes, promoter DNA, TFIIA, TFIIB and TBP. This will be attempted via an international collaborative effort, employing integrated multidisciplinary approach including protein and DNA engineering and their supramolecular assembly combined with chemical cross-linking. This template will be analyzed with and without highly purified, fully recombinant TFIID (sub)complexes through a multi-resolution structural approach with X-ray crystallography, electron microscopy and small angle X-ray scattering techniques. This methodology will be useful to explore interactions of similar complexity involving other chromatin regulatory factors, thereby extending the horizon of transcription research or general multi-protein complex research, which is at the forefront of contemporary molecular biology. The interdisciplinary nature of this project promises to contribute to the applicant’s career development by diversifying and advancing her competencies and refining her skills ultimately guiding her towards a position of independent leadership.