Controlling gene expression is essential to growth, development, and sustained life. A critical control point for regulating gene expression is at the level of transcription. The proper regulation of transcription is essential for maintaining normal pathways of cell growth and differentiation, thereby avoiding the rampant cell proliferation observed in tumors. Transcription of protein encoding genes in eukaryotes is orchestrated by a host of protein factors, including RNA polymerase II, general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), Mediator, coactivators, chromatin remodeling factors, gene-specific transcriptional regulatory proteins (activators and repressors), as well as non-coding RNAs. The underlying goal of our work is to uncover molecular mechanisms governing mammalian RNA polymerase II transcription and its regulation. To this end, we use a combination of biochemistry, molecular biology, molecular genetics, cell-based assays, genomics, and single molecule techniques to investigate mechanisms of mammalian transcriptional regulation.