FIG 8.

Increased imprecise initiation by the tombusvirus replicase from yeast with depleted WW-domain proteins. (A) Scheme of the tombusvirus replicase assay. wt or mutant (TET::RSP5/TET::PRP40/wwm1Δ to deplete WW-domain proteins) yeasts coexpressing FLAG-p33, FLAG-p92, and DI-72 repRNA were used to purify the tombusvirus replicase after solubilization of the membrane fraction of yeast. Note that yeasts were grown for 16 or 64 h after induction of TBSV replication prior to replicase purification. The replicase preparations were programmed with TBSV RI/III (−)RNA that leads to precise initiation [FL, full-length complementary (+)RNA product] and imprecise initiations (ii, internal initiation from cryptic promoter-like sequences; or 3′-TEX, 3′-terminal extension). (B) Denaturing PAGE analysis of the in vitro replicase products. The ratio of imprecise initiation versus precise initiation was calculated.