While many factors are involved in the regulation of gene transcription, each of these must ultimately act to affect the rate at which RNA Polymerase II (Pol II) transcribes a given gene. As this rate is determined at one or more rate-limiting steps in transcription, we expect that a wide array of upstream regulatory process converge at these steps in order to modulate transcription of their target genes. I focus on two putative rate-determining steps present in higher eukaryotes: the processes preceding and including the initiation of gene transcription, including the recruitment of Pol II to gene promoters, and the release of paused Pol II from the promoter-proximal region into the gene body where it can then mediate productive gene transcription. After a general introduction to Pol II pausing and a presentation of novel software I’ve written for studying transcription, the next major section of my dissertation focuses on the pausing factor NELF, which binds Pol II to induce pausing in higher eukaryotes. Using Drosophila melanogaster as a model system, I find that the presence of NELF significantly enhances the regulatory potential of the transcriptional kinase Cdk9 in higher eukaryotes by establishing an efficient system of gene regulation based around NELF and Cdk9. After additional ancillary findings concerning NELF and another factor DSIF in Drosophila, I present an innovative experimental approach for determining the rate-limiting step at individual genes across the human genome. Preliminary findings here show that I can identify distinct sets of genes that are subject to regulatory at distinct points in transcription, laying the groundwork for the discovery of specific upstream regulatory pathways acting at these two discrete rate-determining steps modulating gene expression.