Figure 5.
Effect of ScKti12 mutants on Elongator's U34 modification function. (A) Quantification of modified U34 nucleosides in tRNAs from yeast strains carrying kti12 nucleotide binding pocket mutations. For each mutant, the relative levels of ncm5U, s2U, mcm5U and mcm5s2U are shown. Modified nucleoside signals were normalized using the total uridine content A (modified U)/ A (U), to allow comparison between different samples. ncm5, s2, mcm5 and mcm5s2 are highlighted in red within their chemical structure (top). (B) Zymocin-/γ-toxin- and SUP4-based assays indicative for Elongator function and U34 modification in vivo. WT (KTI12) and Elongator deficient elp3Δ and kti12Δ strains served as growth control for loss-of-function mutants. Ten-fold serial cell dilutions of the strains expressing Kti12 variants with the indicated mutations in the nucleotide binding pocket mutants (see A) were spotted on SD glucose media with 60 μg/ml canavanine, or 5% (v/v) zymocin, lacking adenine (- Ade) or containing 2 % (w/v) galactose (Gal: γ-toxon) instead of 2% (w/v) glucose (Glc: γ-toxoff). For the γ-toxin assay, strains were transformed with vector pLF16 expressing the γ-toxin gene under the galactose inducible GAL1 promoter (68) and cultivated for 3–4 days at 30°C. (C) Kti12-Elongator interaction studies. Yeast strains co-expressing c-myc-tagged Elp1 and HA-tagged Kti12 variants were subjected to immunoprecipitation (IP) using anti-c-myc antibodies. IPs were examined by Western blotting with anti-c-myc (to detect Elp1) and anti-HA antibodies (to detect co-immunoprecipitated Kti12). Western blots of the extracts prior to the IPs (Pre-IP) controlled the input using anti-HA and anti-Cdc19 antibodies, to confirm similar levels of Kti12 and pyruvate kinase (Cdc19) in the different strains. (D) Purified ScKti12 and ScElp1 interact in vitro. Full length GST-ScElp1 successfully bound ScKti12–6xHis in a concentration-dependent manner. Free GST was used as a specificity control. For GST pull-down assay, gels were stained with Coomassie and respective proteins are labeled next to the gel.