Transcription of DNA into mRNA by RNA Polymerase II (Pol II) is a highly dynamic and complex process that requires the concurrent collaboration of many factors. One such factor is Spt5 - a multi-domain transcription elongation factor that acts as a component of all Pol II elongation complexes. It is universally conserved and essential for life, playing a central and ancient role in transcription.Recent structural studies indicate that several of Spt5’s central KOW (Kyprides, Ouzounis, Woese) domains lie in close contact with the dissociable stalk of RNA Polymerase II (subunits Rpb4 and Rpb7) (Bernecky et al., 2017). Both Spt4/5 and Rpb4/7 have been previously implicated in polyadenylation (poly(A)) site choice (Cui et al., 2003; Runner et al., 2008) 3’ end processing of mRNA (Mayer et al., 2012; Runner et al., 2008), mRNA export (Burckin et al., 2005; Farago et al., 2003), and allosteric stabilization of elongating Pol II (Armache et al., 2016; Bernecky et al., 2017). Despite these structural studies that report physical interactions between these proteins, a functional interaction has yet to be revealed. x Here we present evidence that Spt5 and Rpb4/7 collaborate to execute functions relating to 3’ end formation, mRNA export, cotranscriptional chromatin maintenance and R-loop formation. We take a genetic and biochemical approach to address the consequences of disrupting the direct interactions of these proteins, revealing a series of allele-specific genetic interactions between SPT5, RPB4 and RPB7 that point to a functional cooperation throughout transcription elongation and termination. Affinity chromatography of yeast cell extracts using isolated Spt5 KOW domains as bait revealed a large set of KOW-interacting proteins. Many of these were previously reported to interact with Rpb7 (Mosley et al., 2013). These data point to a direct, functional cooperation between Spt5 and Rpb4/7 to regulate the processing and export of mRNA, revealing new roles for the previously ambiguous central domains of Spt5. Chapter 1 summarizes the literature and current gaps in our knowledge with regards to the function of Spt5’s central domains and the Pol II stalk in transcription. Chapter 2 presents a genetic analysis of spt5 and rpb4/7 mutants. We have identified mutations at multiple points throughout the structure formed by Spt5 KOW2-4 and Rpb4/7, including the juncture of KOW3/Rpb7 and KOW4/Rpb7, as well as multiple solvent exposed regions on the surface of this structure. Many of these mutations genetically interact with each other, resulting in synthetic sickness and enhancement of phenotypes. Interestingly, xi mutations in Rpb4 and Rpb7 both share the cryptic transcription phenotype that pervades many known spt5 alleles as well as chromatin remodeling proteins, suggesting that the core structure of Pol II itself evolved to assist in overcoming nucleosomal barriers to transcription. Chapter 3 reports identification of biochemical interactions between Spt5’s central KOW domains and their interacting factors. After establishing that Spt5 KOW2-4 and Rpb4/7 extensively interact genetically, we aim to test the hypothesis that this region functions as a binding platform for tertiary factors. We performed affinity chromatography followed by MudPIT mass spectrometry to identify binding partners of both the KOW2-3 region (K2K3) and the Linker2-KOW4 (L2K4) region of Spt5 (Delahunty, Yates 2007). Supporting the notion that the central KOW domains function in tandem with Rpb4/7, we identified a large number of proteins involved in 3’ end formation, RNA processing and chromatin structure maintenance that overlap with previous studies of Rpb7. Chapter 4 explores the functional relationship between Spt5, Rpb7, Nrd1 and R-loop formation. Together with evidence from previous chapters, we propose a model for the Pol II Stalk region in functioning to maintain R-loop homeostasis by bridging interactions with tertiary factors. Chapter 5 summarizes the main findings of this work and describes the contribution made to our existing knowledge gaps in transcriptional biology.