RNA Polymerase II-mediated transcription is a critical, highly-regulated process aided by protein complexes at distinct steps. One key, rate-limiting step in transcription regulation is release of Pol II from the promoter-proximal pause into productive elongation. Decades of work have uncovered many of the protein factors involved in Pol II pause and release and ascribed functions to them. However, several mechanistic details of the pause to pause release transition have yet to be fully uncovered. Recent high resolution cryo-EM structures derived from in vitro assemblies provide a roadmap of mechanistic hypotheses to test regarding Pol II pause and release in live cells. In this thesis, I describe our work investigating the kinetic behavior of several key factors involved in Pol II elongation using Drosophila melanogaster polytene chromosomes and to a lesser extent, high resolution genomics. By knocking-in Photoactivatable-GFP (PA-GFP) or HaloTag, we optically highlighted and tracked populations of molecules, and observed their recruitment at the induced Hsp70 loci. We found that early in the heat shock response, Pol II and the elongation factor DSIF are not reused for iterative rounds of transcription. Next, we examined the recruitment and dynamics of two important elongation factors, Spt6 and PAF1. We showed that these factors are not recruited to Pol II without the activity of the P-TEFb kinase, surprisingly they show very different binding behaviors despite structural predictions, and finally PAF1 may be dispensable for elongation when nucleosome densities are low. Additionally, we began to probe the steps that Pol II must undergo to transition from a stably paused complex into a mature elongating complex. We observed that NELF dissociates from Pol II as Pol II transcribes into the +1 nucleosome and this corresponds with Spt6 binding. We also began to query the binding and residence of factors involved in transcription initiation and resolving stalled or backtracked Pol II. In summary, our live-cell data provides new insight and key constraints to mechanistic models of transcription regulation.