Structure-activity relationships in Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease.
tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis gamma-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of gamma-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNA(Glu). Fungal gamma-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to gamma-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in gamma-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free gamma-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA(Glu) at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that gamma-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.
Keppetipola
Niroshika
N
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
Jain
Ruchi
R
Meineke
Birthe
B
Diver
Melinda
M
Shuman
Stewart
S
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D055646:Killer Factors, Yeast / Q000737:chemistry* / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D055646:Killer Factors, Yeast; D011994:Recombinant Proteins; C058975:zymocin; D018698:Glutamic Acid; D006639:Histidine; D012343:RNA, Transfer; D001120:Arginine; D012260:Ribonucleases; C049345:anticodon nuclease; D008239:Lysine