FIGURE 5. N-subdomain mutants are defective in EC formation.

WT and mutant T7 RNAPs were incubated with a promoter template (RB3/RB4) in the presence of GTP, ATP, and CTP to form an EC halted at 15 nt or a nucleic acid scaffold (RB5/RB6/RB7) that gives rise to an EC. The complexes were subjected to limited digestion with trypsin, and the products were analyzed by 4 –20% Tris-glycine SDS-PAGE. The sizes of protein molecular weight (MW) markers are shown in the margin.