In eukaryotes, RNA polymerase II (Pol II) is responsible for the transcription of all protein-coding genes, and regulation of its activity is fundamental for cellular homeostasis and the programmed development of multicellular organisms. The transcription process is regulated by the coordinated action of transcription factors (TFs), which interact with each other, Pol II and specific regulatory sequences to modulate distinct rate-limiting steps in the transcription cycle. Identifying the regulatory TFs and the biochemical processes that are controlled by each factor is therefore critical for understanding how transcription is regulated. When studying mechanisms of transcription regulation, inducible systems are an invaluable resource: regulatory processes can be triggered instantaneously, enabling the tracking of ordered mechanistic events. We used Precision Run-On sequencing to examine the genome-wide Heat Shock (HS) response in Drosophila and the function of two key TFs on the immediate transcription activation or repression of all genes regulated by HS. We identified the primary HS responsive genes and the rate-limiting steps that GAGA-Associated Factor (GAF) and Heat Shock Factor (HSF) regulate. We demonstrated that GAF acts before promoter-proximally paused Pol II formation, likely at the step of chromatin opening, and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing, but is critical for the release of paused Pol II into the gene body at a subset of highly-activated genes upon HS induction. Additionally, HSF has no detectable role in the rapid HS-repression of thousands of genes. As a complementary approach, we have selected RNA aptamers to the human HSF1 and HSF2 with the goal of expressing these aptamers in vivo to disrupt specific macromolecular interactions of these TFs. I describe the results and thorough characterization of a successful RNA aptamer selection to HSF1 and HSF2 using our recently developed SELEX technology. I also describe our development and implementation of a set of SELEX performance metrics to evaluate selection success. Our novel SELEX methodology and analysis tools offer a significant improvement over traditional approaches and provides an efficient platform for the performance and analysis of SELEX experiments.