FIGURE 2: Among genes involved in Elongator regulation and tRNA modification, KTI11 and KTI13 also function in EF2 modification. (A) Growth assays in response to zymocin (0.02% [v/v]) or sordarin (9 µg/mL) and diagnostic for tRNA or diphthamide modifciation defects, respectively. Dilutions of cells with indicated genotypes were incubated at 30°C for 3 days. Note, that while all ktiΔ and sit4Δ mutants resist growth inhibition by Elongator-dependent tRNase zymocin, only kti11/dph3Δ and kti13Δ cells are protected (green arrows) against diphthamide-dependent EF inhibitor sordarin. (B) Western blot analysis of total cell extracts from strains with genotypes as in A in order to profile their amounts of total EF2 and unmodified EF2 using anti-EF2(pan) (left panel) and anti-EF2(no diphthamide) antibodies (right panel), respectively. Black asterisks (left & right panels) denote EF2 degradation products, the red asterisk indicates full-length unmodified EF2 (right panel). The anti-Cdc19 antibody (bottom panel) was used as loading control. Note the anti-EF2(no diphthamide) Western blot (right panel) detects unmodified EF2 pools for kti11/dph3Δ and kti13Δ cells indicative for diphthamide defects.

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