Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC.
tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in a eukaryon in the absence of other bacterial or viral proteins. Whereas Streptococcus PrrC is also toxic in yeast, Neisseria and Xanthomonas PrrCs are not. Via analysis of the effects of 118 mutations on EcoPrrC toxicity in yeast, we identified 22 essential residues in the NTPase domain and 11 in the nuclease domain. Overexpressing PrrCs with mutations in the NTPase active site ameliorated the toxicity of wild-type EcoPrrC. Our findings support a model in which EcoPrrC toxicity is contingent on head-to-tail dimerization of the NTPase domains to form two composite NTP phosphohydrolase sites. Comparisons of EcoPrrC activity in a variety of yeast genetic backgrounds, and the rescuing effects of tRNA overexpression, implicate tRNA(Lys(UUU)) as a target of EcoPrrC toxicity in yeast.
Meineke
Birthe
B
Sloan-Kettering Institute, Weill Cornell Medical College, New York, NY 10065, USA.
Schwer
Beate
B
Schaffrath
Raffael
R
Shuman
Stewart
S
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D012357:RNA, Transfer, Lys; D012343:RNA, Transfer; C084965:PrrC protein, E coli; D012260:Ribonucleases; D000409:Alanine