• Molecular Analysisof Saccharomyc escerevisiae RNA Polymerase I Core Factor Complex and its Interaction with Promoter DNA

      Knutson, Bruce; Jackobel, Ashleigh J (2020)
      Gene transcription and protein synthesis are essential molecular processes required for all living organisms. In eukaryotes, messages encoded within DNA are transcribed by three DNA-dependent RNA polymerases (Pols I-III) into ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. General transcription factors (GTFs) help recruit Pols to their appropriate gene promoters as well as facilitate template opening and transcription start site (TSS) selection. In Saccharomyces cerevisiae, the Pol I pre-initiation complex (PIC) is formed by numerous GTFs that include Upstream Activating Factor (UAF), Core Factor (CF), TATA-binding protein (TBP), and Rrn3. This unique set of GTFs engage ribosomal DNA (rDNA) through interactions with regulatory elements of the promoter known as the Upstream Activating Sequence (UAS) and the Core Element (CE). Here, we resolve the cryo-electron microscopy (cryo-EM) structure of CF bound to the rDNA promoter at 3.8Å near-atomic resolution and determine itsDNA binding preferences in which CF preferentially binds to the GC-minor groove. Briefly, our cryo-EM studies reveal that the CF-DNA interaction is mediated by two CF subunits, Rrn7 and Rrn11. We also found that the path of promoter DNA is relatively straight in the Pol I PIC, which is strikingly different from the bent promoters observed in structures of the Pols II/III PICs. We identified three states of CF engagement with promoter DNA (States 1-3) in which CF acts as a ratchet toforceDNA into the active site of the polymerase that facilitates the melting of the double-stranded DNA template in an ATP-independent manner, another unique feature of the Pol I system. Using in vitroDNA binding assays, we have identified a 12 base pair (bp) region within the CE that is necessary and sufficient for CF binding. We have also demonstrated that the human anticancer compound CX-5461 can inhibit yeast cell growth and blocks yeast CF binding to both yeast and human rDNA promoters in vitro. Furthermore, we show that the human Core Promoter Element (CPE) can functionally replace the yeast CE in a position-dependent manner. Together, these results reveal the unique molecular architecture of the Pol I PIC and suggest a conserved sequence-independent binding mechanism of CF with promoter DNA.