Figure 5. Reactivity of wild type and mutant E2~Ub thioester in the presence and absence of RNF4.

The rates of ubiquitin discharge from the E2~Ub to lysine in the presence (black columns) and absence (grey columns) of RNF4, with native RAD6B and UBCH5B, UBCH5Bs6B, as well as with the RAD6Bs5B conversion constructs. E2 was pre-charged with fluorescein-tagged ubiquitin, Ub^FL, and then added to the E2~Ub^FL discharge reaction. E2~Ub^FL discharge reactions contained 50 mM lysine, 10 μM RNF4, and 5 μM E2~Ub^FL. Ub-discharge was monitored by SDS-PAGE, followed by fluorescent imaging, and fluorescence signal densitometry.