FIGURE 6.

Elongin A mutations differentially affect assembly with CUL5 and recruitment to sites of microirradiation. A, schematic representation of wild type Elongin A and mutants analyzed in this study and their abilities to bind Elongins B and C and to stimulate Pol II elongation or support Rpb1 ubiquitylation in vitro. B, FRET efficiencies in U2OS cells (non-irradiated or 5 min after microirradiation) expressing wild type or mutant Halo-Elongin A and mCherry-CUL5. Values represent average ± S.E. (error bars) (n ≥ 12); *, p ≤ 0.005. C, scatter plot showing Halo-Elongin A relative pixel intensity, measured before acceptor photobleaching, as a function of FRET efficiency in individual cells. D, kinetics of recruitment (n ≥ 12) to microirradiated regions of wild type and mutant Halo-Elongin A. Cells were imaged every second, and intensity values were binned over 5-s intervals. Microirradiation was initiated at time = 0 s.