Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

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Dysregulation of RNA polymerase I (Pol I) is a hallmark of many cancers and several developmental disorders. Pol I is a multi‐subunit complex that synthesizes ribosomal RNA (rRNA), the integral structural and catalytic component of the ribosome. A key yeast Pol I subcomplex that functions in nearly every step of the transcription cycle is the Rpa34/Rpa49 heterodimer. Recent studies with the heterodimer's human counterpart have shown species specific differences in terms of the heterodimer’s importance for cell growth and functional domain requirements. To further explore these differences, we used the yeast model system to show that similarly to humans, the yeast heterodimer subunits exhibit codependent expression. We further analyzed this codependence in terms of specific heterodimer domain requirements and the level of gene expression the codependence occurs. We found that Rpa34/Rpa49 codependence occurs at the post translational level in a manner dependent on Rpa49’s dimerization domain. Overall, our findings suggest that the codependent expression of yeast and human Rpa34/Rpa49 subcomplexes is an evolutionarily conserved feature that may apply to other Pol I, II, and III subcomplexes.