The Mediator is a complex protein machine, consisting of 25 subunits with a total molecular weight of 1.2 MDa. This complex is crucially involved in eukaryotic gene transcription.  Consequently, determining Mediator’s structure and the interactions between its subunits is important to understanding its role in gene regulation.  Thus far, attempts to purify and crystallize the component proteins of the human Mediator have been unsuccessful, as the Mediator subunits have uncommonly long unstructured domains. Studies on protein-protein interactions in the yeast Mediator are incomplete and have required extensive labor and costs.  We used Alpha Screen technology, combined with cell-free protein expression, to rapidly map binary interactions between the Mediator proteins constituting the tail domain. Co-expression of the proteins allows co-folding of the subunits and enables us to successfully reconstitute interactions in vitro. Based on the collected data from 64 protein pairs, we were able to estimate the architecture of the tail domain and determine the key subunits in its assembly.

Further, we would like to expand the screening to the entire Mediator complex, and measure ternary and higher-order interactions among the subunits. In order to handle the resulting increase in complexity and the number of combinations (>2,000), we will implement a novel microfluidic platform for high-throughput screening of interactions. The measurement technique, developed in Stephen Quake’s laboratory is called MITOMI, for mechanically induced trapping of molecular interactions. It uses microfluidic valve technology to trap interacting proteins in the reaction chambers and provides simultaneous measurement of hundreds of interactions. Taking advantage of on-chip cell-free protein expression, the target proteins’ DNA sequences can be mixed with the protein-expression lysate in the reaction chambers, in any desired combination.  In this way, we hope to quantify the full interaction map of the Mediator complex in a single experiment.