Fig. 2.

Determining the molecular architecture of human RNAPII. Top, data gathering. Comparative models of the H-RNAPII subunits were obtained from the ModBase database (54). A density map of H-RNAPII at 20-Å resolution (50) was obtained from the EM data bank (56). Proteomics data for S. cerevisiae RNAPII subunits were obtained from BioGRID (Table III) (55). All pairwise direct interactions are visualized in a single graph with solid edges, and each pulldown experiment is presented as a separate graph with dashed edges to indicate the missing underlying binary interaction network. Pulldowns Rpb1-Rpb2-Rpb3-Rpb4-Rpb5-Rpb8 and Rpb1-Rpb2-Rpb3-Rpb8-Rpb10 are missing some edges for clarity. Gray edges indicate interactions present in BioGRID but not in the yeast RNAPII crystallographic structure. Middle, scoring. The scoring function is the sum of the distance (illustrated between Rpb4 and Rpb7), connectivity (illustrated between Rpb1, Rpb2, Rpb3, Rpb8 and Rpb10), EM quality-of-fit (illustrated between the H-RNAPII density map and Rpb1), and geometric complementarity (illustrated between Rpb4 and Rpb7) restraints. Bottom, optimization. The configuration of the subunits in H-RNAPII was optimized using an extension of the divide-and-conquer MultiFit protocol to incorporate proteomics-based restraints. The optimization procedure resulted in a single model that satisfied all of the input restraints.