PICen

Precise control of eukaryotic gene expression at the transcriptional level is essential to orchestrate many vital cellular processes including homeostasis, differentiation and cell death. Our project aims to discover the molecular mechanisms that coordinate this process during the initiation step of transcription. We propose to investigate the biochemical composition and the structure/function relationship of transcription initiation complexes formed in vivo by taking an integrative approach involving advanced protein purification, cryo-electron microscopy, crosslinking-coupled mass-spectrometry, genome-wide promoter mapping and transcription profiling methods. The assembly of the transcription preinitiation complex (PIC), composed of general factors (GTFs) and the Pol II enzyme, on gene promoters is a key regulatory step that selects the gene to be expressed. PIC assembly on a TATA-box containing promoter has been reconstituted in vitro and involves the interaction of the TATA Binding Protein (TBP) with its cognate promoter sequence, the subsequent binding of the GTFs, TFIIA and TFIIB, that recruit Pol II with the remaining GTFs: TFIIE, TFIIF and TFIIH. Close to atomic resolution structures of this in vitro reconstituted PIC have been determined recently. However, in vivo TBP associates with 13 (14 in yeast) TBP-associated factors (TAFs) and forms the transcription factor IID (TFIID) that not only nucleates PIC formation, but has been suggested to function also as a transcriptional co-activator by interacting with transcription activators. Importantly, the structure of TFIID-containing PICs is not known, leaving open the possibility that the composition and architecture of in vivo assembled PICs may differ from that reconstituted in vitro. Recently we made important progress in the purification of large amounts of endogenous TFIID complexes and native DNA-bound PICs from Komagataella phaffii cells. To better understand the role of native TFIID in endogenous PIC formation and consequent transcription initiation we have the following aims: 1) Characterize how TFIID binds to promoter DNA in vitro and how TBP participates in this process 2) Characterize the composition and structure of endogenous TFIID-containing PICs 3) Describe the subunit interaction landscape of promoter DNA associated native TFIID-containing PICs 4) Genome-wide identification of transcriptionally active promoters bound by Tafs, TBP, Pol II and GTFs to define different promoter classes occupied by distinct TFIID-containing PICs Thus, in our collaborative project, based on long-standing interactions between the partners, we will uncover the specificity of the TFIID/TBP-promoter DNA interactions, how TBP behaves during transcription initiation, and whether TBP detaches form TFIID during in vitro promoter binding. Our proposal will also describe the subunit composition and the molecular structure of promoter bound TFIID-containing initiation complexes assembled in vivo. Our novel purification protocol of native TFIID and endogenous PICs opens the possibility to determine the structure of native transcription initiation complexes together with their interaction landscape. This information combined with genome-wide TAF, TBP, GTF and Pol II occupancy and gene expression profiling data sets will identify distinct sets of TFIID-containing PIC assemblies on different promoter classes that are involved in transcription initiation., our preliminary data indicate that native PICs differ significantly from in vitro reconstituted PIC assembly. Thus, our results will shed new light on the native transcription initiation process by determining the composition and structure of key TFIID-containing initiation complexes formed in live cells.