Substrate specificity and mutational analysis of Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

Abstract

tRNA anticodon damage inflicted by the Kluyveromyces lactis g-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. g-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 39 of the wobble base of tRNA Glu(UUC) to generate a break with 29,39-cyclic phosphate and 59-OH ends. Recombinant g-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA Glu . A single 29-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to g-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 59 of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting g-toxin's cleavage activity. Whereas either the 59 half or the 39 half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that g-toxin requires an A-form helical conformation of the anticodon stem. We purified g-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that g-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151).

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

“…Yet, the anticodon nuclease subunits PaOrf2 and g-toxin are seemingly unrelated (Klassen et al 2004). An expanded comparison to putative ribotoxins encoded by related linear plasmids from P. inositovora and D. robertsiae led us to identify a small number of conserved residues, two of which-a glutamate and a histidine-are known to be essential for g-toxin activity (Keppetipola et al 2009;Jain et al 2011). We find that the corresponding Glu and His residues of PaOrf2 are essential for its toxicity in vivo.…”

“…2). Thus, five of the eight residues targeted, including those directly involved in the proposed transesterification mechanism of g-toxin (Jain et al 2011), were essential for PaOrf2 function, suggesting similarities in the g-toxin and PaOrf2 active sites.…”

“…2). The equivalents of PaOrf2 Glu9 and His287 in g-toxin (Glu9 and His209) are imputed to be active site residues, at which even conservative substitutions led to a loss of toxicity in vivo and ablation of anticodon RNase activity in vitro (Jain et al 2011). Here, we substituted PaOrf2 Glu9 with glutamine and aspartate and His287 with glutamine and asparagine and checked the effect on the in vivo activity of PaOrf2.…”

“…Alignment of the protein sequences in search of a structural or evolutionary connection between PaOrf2 and g-toxin is now aided by the identification of two other putative plasmid-encoded fungal ribotoxins (PiT of Pichia inositovora and DrT of Debaryomyces robertsiae) and by the results of an extensive mutational analysis of g-toxin that identified an ensemble of amino acids required for toxicity in vivo and anticodon nuclease activity in vitro (Jablonowski et al 2006;Keppetipola et al 2009;Jain et al 2011).…”

“…1). Two of these correspond to g-toxin Glu9 and His209, which are essential for toxicity and are the imputed general acid-base catalysts of RNA transesterification at the wobble uridine (Jablonowski et al 2006;Jain et al 2011). Here, we used the alignment in Figure 1 to guide a mutational analysis of PaOrf2 and thereby identified five side chains that are critical for toxicity in vivo, including the conserved glutamate and histidine residues.…”