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Ubiquitination of Rpb1 by Rpf1 in vitro. Rpb1 was translated in vitro in rabbit reticulocyte lysate in the presence of [35S]-methionine. Purified hect E3 proteins (human E6-AP, human Rpf1 [WW-hect; amino acids 193 to 901], yeast Rsp5, and the mutant of Rsp5 with a change of the active-site Cys to Ala [C-A]) were incubated as indicated with Rpb1 in the presence of ATP, ubiquitin, E1 enzyme, and E2 enzyme (Arabidopsis thaliana Ubc8) as previously described (18).
