tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Introduction The yeast secretes a tRNA anticodon nuclease toxin encoded by a resident cytoplasmic linear DNA “killer” plasmid (Satwika et al. 2012 The secreted form of the ribotoxin is a complex of PaOrf1 and PaOrf2 subunits that arrests growth of the non-self Posaconazole yeast (Fig. 1A). The chitin-binding PaOrf1 subunit interacts with the target cell surface to effect the delivery of Posaconazole the PaOrf2 subunit – the toxin PaT – into the cytoplasm of the target cell. PaT elicits toxicity via cleavage of the anticodon loop of tRNAGln(UUG) (Fig. 1B) thereby depleting the functional pool of this isoacceptor (Klassen et al. 2008). To prevent self-killing either by autocrine uptake of secreted toxin or by residual free cytoplasmic PaT the killer plasmid encodes an immunity factor ImmPaT (the PaOrf4 protein) that protects via an unknown mechanism (Paluszynski et al. 2007 Fig. 1 PaT is toxic and incises a synthetic tRNAGln stem-loop at a unique site 3′ of the wobble uridine The yeast has an analogous system of self-nonself discrimination via the secreted toxin zymocin the γ-toxin subunit of which is an anticodon nuclease that targets tRNAGlu(UUC) (Lu et al. 2005 In addition to their primary targets PaT and γ-toxin can also cleave albeit to a lesser extent the two other yeast tRNAs that have an mcm5s2U wobble base: tRNAGlu and tRNALys for PaT; tRNAGln and tRNALys for γ-toxin (Lu et al. 2005 Klassen et al. 2008 Whereas γ-toxin is strictly dependent on the wobble mcm5 modification for its ribotoxicity PaT is not. It is noteworthy that despite their tRNA substrate overlap PaT has no apparent primary structure similarity to Posaconazole γ-toxin. Nor does the PaT primary structure resemble any of the exemplary bacterial tRNA anticodon nucleases: PrrC (Blanga-Kanfi et al. 2006 colicin E5 (Ogawa et al. 1999 colicin D (Tomita et al. 2000 or VapC (Winther and Gerdes 2011 Indeed PaT has no similarity to any known nucleases or phosphotransferases. Its sole retrievable homolog is a plasmid-encoded toxin of unknown target specificity from the yeast (Klassen et al. 2004 The fungal ribotoxins are a fertile area for exploring the mechanisms structures and evolution of eukaryal tRNA restriction enzymes which are of heightened interest in light of recent discoveries of tRNA restriction as a general eukaryal response to cellular stress (Thompson and Parker 2009 Saikia et al. 2012 γ-toxin has been studied genetically and biochemically. It has been mutagenized extensively and its Mouse monoclonal to CD34 cleavage mechanism and specificity have been defined using native tRNAs and synthetic RNA substrates that mimic the anticodon stem-loop of tRNAGlu (Lu et al. 2005 2008 Keppetipola et al. 2009 Jain et al. 2011 Yet γ-toxin has so far eluded a structure determination. By comparison knowledge of PaT is sparse. Here we purify biologically active homogenous PaT characterize its Posaconazole anticodon nuclease activity determine its atomic structure by X-ray crystallography and Posaconazole illuminate structure-activity relationships. We also purify ImmPaT and determine how it interdicts self-killing. Results Purification of bioactive PaT The bioactivity of PaT can be assayed by galactose-induced intracellular expression in of a version of the PaT protein that lacks the predicted N-terminal signal peptide and instead has the N-terminal sequence MNPTTCLNE in which a new initiating methionine is appended to the native asparagine (Klassen et al. 2004 (Fig. 1C). Here we found that toxicity was unaffected when the translation start was shifted one residue to the left by encoding the N-terminus MGNPTTCLNE in which the glycine derives from the native PaT (Fig. 1C). In this context the N-terminal Posaconazole methionine will be removed by yeast methionine aminopeptidase (Moerschell et al. 1990 By phasing the start site to the right in single amino acid increments we determined that replacing Asn2 with methionine did not affect the toxicity of PaT as gauged by inhibition of cell growth on medium containing galactose (Fig. 1C). By contrast deleting Asn2 and replacing Pro3 with methionine or deleting Asn2-Pro3 and replacing Thr4 with methionine abolished PaT toxicity and allowed growth on galactose (Fig. 1C). Based on these findings we elected to purify and characterize the.