Fig. 2.

Phenotypic characterization of Kti12 K14L mutation and elongated linker in yeast. a Phylogenetic tree based on Kti12 sequence alignment displays distribution of different residues at position 14 among species of different divisions [Ascomycota (orange)/Basidiomycota (green)]. Kti12 proteins carrying L14 and long linker (purple squares) belong to Eurothiomycetes. Multiple sequence alignment of Kti12 proteins from 25 model fungi species was performed using JalView software (Waterhouse et al. 2009) and Muscle server (Edgar 2004). The phylogenetic tree was built using the neighbor-Joining method. Individual branch lengths are shown above the branches and indicate the number of amino acid substitutions per site. The fraction of replicate trees in which the associated taxa clustered together in the bootstrap test [1000 replicates (Felsenstein 1985)] are shown beneath the branching points. The evolutionary distances used to calculate the tree were calculated using the Poisson correction method with the pairwise deletion of ambiguous positions. Final dataset contained 610 positions. All evolutionary analyses were conducted in MEGA X (Kumar et al. 2018). The tree was visualized using iTOL (Letunic and Bork 2007). b Zymocin assay assessing performance of the Elongator complex and presence of U₃₄ modifications. WT yeast strain (ScKTI12) served as a growth control, Scelp3Δ and Sckti12Δ pose negative controls. Tenfold dilutions were spotted on YEPD media with the addition of 50% Zymocin or YNB media replacing arginine by 40 µg/ml canavanine or lacking adenine (-adenine). In the different strains KTI12 was mutated (K14A/L), parts or the whole gene were exchanged with the corresponding sequences from Aspergillus niger (An knot + linker, AnKTI12) or parts of it were deleted (marked with ∆ and the deleted residues in brackets). Sckti12-NTD codes for the first 184 amino acids