Mechanism of TBP delivery to gene promoters by the SAGA co-activator – SAGA-TBP

Transcription initiation is an essential step in gene regulation, which allows organisms to adapt to external changes. To initiate transcription, the TATA-box binding protein TBP has to be delivered in a highly controlled way to promoters of protein encoding genes. The interaction of TBP with the promoter initiates the assembly of a Pre-Initiation Complex (PIC) over the transcription start site (TSS) to start mRNA synthesis. Understanding TBP delivery, turnover and dissociation from gene promoters is therefore of fundamental importance. However, despite decades of research, we are still lacking a quantitative and dynamic description of TBP behavior. The TFIID and SAGA complexes maintain TBP in an inactive state and are essential for the controlled delivery of TBP, at the correct TSS and the right time. The first task of our project aims at combining biochemical assays, yeast genetics, fluorescence measurements and single particle cryo electron microscopy (cryo-EM) in order to identify key functional intermediates that will describe how TBP is released from SAGA by TFIIA and delivered onto promoter DNA.
TFIID and SAGA share a similar molecular device to deliver TBP, but they have distinct roles in gene expression. Transcriptomics and ChIP-seq chromatin profiling indicate that constitutively expressed house-keeping genes rely on TFIID, whereas regulated stress-induced genes are SAGA-dependent. This dichotomous view has been challenged by studies using conditional alleles and nascent transcriptomics showing that SAGA acts on the expression of most genes. To date, the molecular components that determine whether a gene requires SAGA, TFIID, or both, for TBP loading remain unknown. Our second task aims at exploring the selectivity of SAGA for promoters containing a consensus TATA-box, as opposed to TFIID, which can also recognize TATA-less promoters. We will analyze SAGA-dependent TBP delivery on a large variety of promoters by in vitro EMSA, FRET and FCCS methods and in vivo time-resolved crosslinking ChIP approaches that will correlate the sequence properties of endogenous core promoters with TBP turnover. We will generate TFIID/SAGA hybrids to assess whether the TBP binding subunits provide the specificity for TATA-box recognition. Our third task will explore the role of Mot1 in SAGA-dependent TBP release. Mot1 interacts genetically with Spt3 and has been suggested to regulate its promoter selectivity. We will analyze the effect of Mot1 on SAGA-dependent TBP delivery and study whether a stable complex can be formed between Mot1 and SAGA in order to analyze its cryo-EM structure.
SAGA is believed to be recruited to gene promoters by acidic transcriptional activators and its large Tra1 subunit is considered as a major in vitro and in vivo target for activator binding. However, neither the activator binding interfaces nor the allosteric regulatory mechanisms relating activator binding with the other SAGA functions have been described so far at the structural level. Moreover, the essence of transcriptional co-activators, such as SAGA, is to operate in a chromatin environment that needs to be modified and remodeled in order to overcome the repressive action of nucleosomes on transcription initiation. The functional interplay between SAGA and the chromatin organization of the gene promoter has not been addressed so far at the structural level. Our fourth task will be devoted to these aspects and we will analyze the structure of activator-bound SAGA, study the effect of activator and chromatin binding on TBP delivery, and explore the long-range effect of SAGA, which bridges upstream activating sequences to the TSS.
We expect that our results will shed new light on the mechanism of TBP delivery, the recruitment mode of SAGA by activators, the role of chromatin structure, and how TFIID and SAGA share the workload of gene transcription.