Figure 5.

Minimal Machinery for Stalled RNC Ubiquitination

(A) Ubiquitination reactions of ³⁵S-labeled 80S F-VHP-β RNCs with the indicated components comparing fraction 2 of the ribosome salt wash with purified Listerin at 0.3 nM, a concentration equivalent to that found in fraction 2.

(B) ³⁵S-labeled 80S F-VHP-β RNCs were incubated with 75 nM E1, 250 nM UbcH5a, 10 μM ubiquitin, 50 nM splitting factors, 250 nM eIF6, 2.4 nM Listerin, and energy. Aliquots were removed at the indicated time points and analyzed by SDS-PAGE and autoradiography.

(C) Ubiquitination reactions of ³⁵S-labeled 80S F-VHP-β RNCs with the indicated purified factors (Listerin at 12 nM). One reaction was pretreated with RNase (lane 6) before the reaction. Shown are the autoradiograph (top) and Coomassie stain (bottom) of the same gel. (See also Figure S4)