Elucidating the Molecular Mechanism of Divergent Transcription

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Project Summary The goal of this proposal is to elucidate the molecular mechanism of eukaryotic divergent transcription at bidirectional promoters. Divergent transcription, defined as sense and antisense transcripts generated from a common promoter, offers an additional mode of transcriptional regulation. This phenomenon has been observed to be nearly ubiquitous and necessary for cell survival. Strand-specific sequencing methods such as global run on sequencing and nascent elongation transcription sequencing have estimated that over 70% of active promoters from yeast to humans exhibit divergent transcription. This is likely an underestimate as divergent transcript pairs have been shown to be regulated and thus appear unidirectional. Additionally, of the divergent transcript pairs, knockdown of non-coding antisense transcripts results in improper lung, heart, and brain development. The current model for divergent transcription is that an active promoter generates a transcription permissible state where a single Med-PIC assembles, releases a single pol II which produces a sense or antisense transcript and then disassembles, thereby clearing space for assembly of another single PIC to repeat the process. This model is supported by previous structural studies of Med-PIC that utilized a shortened DNA template lacking a complete and endogenous complement of upstream activating sequences (UAS) or in the absence of activator proteins. In my initial study, I investigated the assembly of the PIC on a natural bidirectional promoter by utilizing a DNA template comprising an entire nucleosome-free promoter region (NFR) with UAS(s) and flanking core promoters. Importantly, this promoter architecture is where divergent transcription typically occurs. Interestingly, I observed that two PICs dimerize in vitro via the coactivator Mediator, hereinafter called dMed-PIC in an activator protein dependent manner. In Aim 1, I will resolve a structurally comprehensive view of dMed-PIC using a novel and hybrid approach of cryo-electron microscopy single particle analysis (cryo-EM SPA) and cryo-electron tomography (cryo-ET). I will determine the assembly pathway towards divergent transcription and thereby reveal possible regulatory mechanisms by solving the structure of dMed-PIC. In Aim 2, I will investigate transcription by dMed-PIC in vitro by yeast whole cell extract transcription assays. These proposed experiments will use technically innovative approaches to advance our understanding of the novel dMed-PIC and its function in divergent transcription.