title: Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination.
abstract: tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which, in turn, results in growth arrest or cell death. The eukaryal ribotoxin PaT secreted by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNA(Gln(UUG)). We find that recombinant PaT incises a synthetic tRNA(Gln(UUG)) stem-loop RNA by transesterification at a single site 3' of the wobble uridine, yielding 2',3'-cyclic phosphate and 5'-OH ends. Incision is suppressed by replacement of the wobble nucleobase with adenine or guanine. The crystal structure of PaT reveals a distinctive fold and active site, essential components of which are demonstrated by mutagenesis. Pichia acaciae evades self-toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and blocks nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Anupam K
authors.initials: AK
authors.lastname: Chakravarty
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Radhika
authors.initials: R
authors.lastname: Jalan
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA. Electronic address: s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D056265:Microbial Interactions; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013045:Species Specificity
chemical_list: D005656:Fungal Proteins; D009183:Mycotoxins; D012343:RNA, Transfer; D004722:Endoribonucleases
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