FIG 7.

Pre-steady-state single-encounter ubiquitylation reactions allow the estimation of the individual rates of ubiquitin transfer from Ube2R2 to substrate-modified ubiquitins bound to SCF, indicating that the rate of R54D ubiquitin transfer is substantially higher in the presence of D143K Ube2R2 than in the presence of WT Ube2R2. (a) Time course showing the stepwise transfer of R54D ubiquitin to SCF-bound substrate in the presence of WT Ube2R2. S, substrate. (b) Same as in panel a, except with D143K Ube2R2. (c) Graph plotting either the disappearance of monoubiquitylated substrate (above) or the appearance of diubiquitylated product (below) over time. Error bars represent the standard errors of measurements. (d) Same as in panel c, except with D143K Ube2R2. (e) Schematic showing the rates of ubiquitin transfer to substrate as well as the rates of substrate and product dissociation from SCF with WT Ube2R2 and R54D acceptor ubiquitin. (f) Same as in panel e, except with D143K Ube2R2.