Gene transcription is an exquisitely regulated phenomenon in which multiple layers of protein-protein interactions participate in the integration of the signal. The mechanism is mediated through interaction of transcription factors with a large array of regulatory proteins. “Activators” and “repressors” binding DNA upstream of the transcription factors provide positive or negative signals.  The Mediator complex is an adaptor protein that regulates the transcription of almost every eukaryotic protein-coding gene. With more than 25 subunits and 1.2 MDa in size, this large adaptor is believed to integrate the influence of multiple transcription factors, activators as well as repressors and transduce these signals to the RNA Polymerase II. Despite its critical importance, the mechanism of Mediator assembly and its interactions with nuclear components such as transcription factors and DNA are poorly understood. Here, we combine in vitro protein expression with a nanobead proximity assay and single molecule spectroscopy to systematically analyze these protein-protein and protein-DNA interactions. We construct a comprehensive map of interactions among the Mediator subunits that predicts their spatial localization in the complex. These predictions find support in the composition of Mediator and its subcomplexes found in vivo. We also discovered multiple DNA binding sites that map to the surface of the Mediator model, indicating that the DNA may wrap around the complex and regulate its interaction with transcription factors. To test this idea, we analyzed the binding of 43 human transcription factors from different families to the Mediator tail domain and observed the presence of specific binding sites defined by multiple subunits. Remarkably, our model indicates that such binding sites flank the DNA binding groove on the surface of the complex strongly suggesting that interaction between DNA and the Mediator complex can define “hotspots” for binding of transcription factors.