A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops.
Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs(Arg) including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2',3'-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs(Arg), the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA(Arg)-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs(Arg), which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.
Tomita
K
K
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Ogawa
T
T
Uozumi
T
T
Watanabe
K
K
Masaki
H
H
D000926:Anticodon; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003087:Colicins / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012347:RNA, Transfer, Arg / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism* / Q000494:pharmacology
D000926:Anticodon; D003087:Colicins; D012329:RNA, Bacterial; D012347:RNA, Transfer, Arg; D012260:Ribonucleases