STRUCTURAL BASIS OF A TRANSCRIPTION PRE-INITIATION COMPLEX ON A DIVERGENT PROMOTER
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Biochemistry, Biophysics, and Structural Biology
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Divergent transcription
Mediator
promoter-enhancer looping
RNA polymerase II pre-initiation complex
Saccharomyces cerevisiae
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Abstract
While a long history of molecular biology research has been directed toward the deciphering the fundamental mechanisms of life, a structural understanding of eukaryotic transcriptional activation has remained a longstanding goal in biology. Key observations in early transcriptional activation of RNA Polymerase II transcription, have highlighted the role of sequence specific transcription factors (ssTFs) in the recruitment of Mediator and the transcription machinery to transcriptional enhancers. Observations of bidirectional transcription and stable divergent pre-initiation complex (PIC) formation relative such loci point to the existence of a previously uncharacterized ssTF- and Mediator-dependent intermediate. Through characterization of such assembly intermediates on an endogenous divergently transcribed loci, we have identified common structural intermediates and characterized them by cryo-electron microscopy (CryoEM). The resulting architecture reveals how two distinct Mediator bound PICs form a dimer through the Mediator Tail module, induced by an activator protein localized near the dimerization interface. The Tail dimer is associated with ~80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Additionally, cryo-electron tomography was used to visualize the progress of PIC assembly on the two core promoter regions, providing direct evidence for the role of the Tail dimer in supporting divergent transcription. Altogether, these results provide a structural basis for ssTF- and Mediator-dependent bidirectional transcription activation, the proposed innate state of transcriptional activation, and provide a first-ever visualization of recruitment-based transcription activation through the Mediator Complex.