UBC Theses and Dissertations
Structural characterization of the yeast SAGA complex Vasyliuk, Diana
Transcription initiation is a major regulatory step in eukaryotic gene expression. Packaging of DNA into chromatin serves as a barrier against transcription, thus various chromatin modifiers are required to make chromatin structure less compact and more accessible for transcriptional machinery. Spt-Ada-Gcn5-Acetyltansferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Advances in cryo-EM technologies have enabled the determination of the high-resolution structures of the large Tra1 subunit and, more recently, the Tra1 and the core modules in the context of full yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to structural investigations of SAGA. Here, we report a workflow for preparing high quality SAGA cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.
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