Figure 2.
Uncoupling Ribosome Splitting and Ubiquitination Activities
(A) Coomassie-stained gels of fractions from a high salt extraction of native reticulocyte ribosomes (left) and purified splitting factors (right).
(B) Purified 35S-labeled 80S F-VHP-β RNCs were subjected to ubiquitination assays with the indicated components, and the total reaction products were analyzed by autoradiography.
(C) Drop-off assay to monitor ribosome splitting using purified 35S-labeled F-β RNCs. The total reaction (T), supernatant (S), and pellet (P) fractions were analyzed by autoradiography, and the relative amount of F-β-tRNA in the supernatant was quantified (% drop off).
(D) Purified 35S-labeled 80S F-VHP-β RNCs were subjected to ubiquitination assays with the indicated components, and the total reaction products analyzed by autoradiography. The unmodified substrate (F-V-β-tRNA) and poly-ubiquitinated (poly-Ub) products are indicated. (See also Figure S2.)