RNA Polymerase II (RNAPII) is responsible for the transcription of protein coding genes and is tightly regulated to ensure proper gene expression in the cell. The levels of regulation can range from assembly and nuclear import of the complex itself to direct phosphorylation of the polymerase and its associated factors. Given that much of the foundational work on this topic has been performed in yeast, many questions remain regarding how this process has evolved in multi-cellular organisms. In this dissertation, I explore three important facets of RNAPII regulation, each involving a different protein complex. First, I identify INTS15 as a novel subunit of the Integrator Complex, a metazoan-specific transcriptional regulator involved in many different aspects of gene regulation. Using a multi-modal approach combining proteomics, genomics, and machine-learning, I characterize INTS15’s biochemical role in Integrator architecture and demonstrate its importance in transcriptional regulation. Next, I characterize an additional non-canonical PP2A complex involved in transcriptional regulation. Recent studies have shown that the PP2A scaffold isoform, PPP2R1A, complexes with Integrator and directly modulates RNAPII phosphorylation to negatively regulate pause-release. In this work, I characterize a second non-canonical PP2A complex housing the alternative scaffold isoform, PPP2R1B. I demonstrate that PPP2R1B forms a stable complex with RNAPII and is essential for proper recruitment of RNAPII during transcription. Finally, I define a novel role for the small GTPases, GPN1 and GPN3, in facilitating transcriptional initiation. GPN1 and GPN3 have previously been demonstrated to be essential for assembling RNAPII in the cytoplasm, and consequently, importing RNAPII into the nucleus. In this work, I demonstrate that GPN1 and GPN3 associate with RNAPII at chromatin and are recruited to promoters to facilitate transcriptional initiation. Together, these findings expand our knowledge of RNAPII regulation, revealing new proteins and protein complexes that contribute to its assembly, recruitment, and transcriptional mechanism.