Use of a Yeast TRNase Killer Toxin to Diagnose Kti12 Motifs Required for TRNA Modification by Elongator

Abstract

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex fromKluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin's action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g. nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Fungal Kti12 proteins display unusual linker regions and unique ATPase p-loops

“…The entire AnKTI12 gene from Aspergillus niger was unable to replace the function of the ScKt12, which might occur due to specific-interaction interfaces with the Elongator complex or other binding partners. Our data illustrate that the linker region in Kti12 is as important for the tRNA-modification activity of Elongator as the N-terminal ATPase domain or the C-terminal tRNA-binding domain (Schaffrath et al 2017 ).…”

“…2 a). Inactivating Kti12 mutations would most likely also compromise Elongator’s tRNA modification activity (Schaffrath et al 2017 ) and reduce the selective pressure to preserve a functional Elongator complex. Nonetheless, no reports indicate malfunctioning of Elongator or other tRNA modification pathways in Eurotiomycetes .…”

“…Kti12 harbors two domains, namely an N-terminal ATPase domain and a C-terminal tRNA-binding domain, both connected by a flexible linker region (Fig. 1 a) (Schaffrath et al 2017 ; Krutyhołowa et al 2019a ). Kti12 shows a very high overall sequence and structural similarity to O-phosphoseryl tRNA Sec kinase (PSTK), a kinase responsible for phosphorylation of Ser-tRNA Sec (Sherrer et al 2008 ).…”