STRUCTURAL INSIGHTS INTO RNA POLYMERASE III PRE-INITIATION COMPLEX AND THE PIONEER ROLE OF OCT1

Despite the central role of small non-coding RNAs in RNA metabolism and gene regulation, the mechanisms by which RNA polymerase III (Pol III) is selectively recruited to snRNA promoters remain poorly understood. This is particularly notable because Pol II- and Pol III-transcribed snRNA promoters share highly similar architectures and rely on the same core transcription factor, SNAPc. How a single complex enables mutually exclusive recruitment of two distinct RNA polymerases remains an unresolved question. To address this, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of the human Pol III pre-initiation complex (PIC) assembled on the U6 snRNA promoter. Captured in melting and open DNA states (3.2–4.2 Å resolution), these structures reveal distinct SNAPc conformations when engaging Pol III versus Pol II. Through integrative modeling and crosslinking mass spectrometry, we structurally localized the SNAPC2 and SNAPC5 subunits near promoter DNA. These subunits had previously been proposed to act as an autoinhibitory module, and our work provides a structural framework supporting this regulatory role. Together, these findings uncover the structural basis of polymerase specificity and reveal critical differences between human and yeast Pol III PICs, suggesting evolved regulatory mechanisms in higher eukaryotes. In parallel, we investigated OCT1 (POU2F1), a transcription factor known to bind distal sequence elements (DSEs) upstream of Class III Pol III promoters. Although widely expressed and implicated in gene regulation, OCT1’s potential role as a pioneer transcription factor has not been structurally defined. Motivated by its consistent presence at Class III promoters, we used cryo-EM to determine the structure of OCT1’s DNA-binding domain bound to nucleosomes. The data reveal three distinct binding modes: one sequence-specific interaction at the DSE and two non-specific contacts at superhelical locations that engage and remodel histone H4 tails. These interactions reposition histone tails and displace linker histone H1, resulting in chromatin destabilization and increased DNA accessibility. Altogether, this thesis provides structural and mechanistic insights into transcription initiation by Pol III at snRNA promoters and uncovers OCT1 as a chromatin-interacting factor with pioneer-like activity. These findings advance our understanding of polymerase recruitment and chromatin regulation in non-coding RNA gene expression.