A Cytotoxic Ribonuclease Which Specifically Cleaves Four Isoaccepting Arginine tRNAs at Their Anticodon Loops

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 15; pp. 8278 - 8283
• Main Authors: Tomita, Kozo, Ogawa, Tetsuhiro, Uozumi, Takeshi, Watanabe, Kimitsuna, Masaki, Haruhiko
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 18-07-2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Amino acids; Anticodon; Base Sequence; Binding Sites; Biochemistry; Biological Sciences; Catalysis; Catalysts; Cells; Colicin D; Colicin E5; Colicins - metabolism; Colicins - pharmacology; DNA probes; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Immunity; Molecular Sequence Data; Molecules; Mutagenesis; Nucleic Acid Conformation; Phosphatases; Plasmids; Proteins; ribonucleases; Ribonucleases - metabolism; Ribonucleases - pharmacology; Ribonucleic acid; Ribosomes; RNA; RNA, Bacterial - chemistry; RNA, Bacterial - isolation & purification; RNA, Bacterial - metabolism; RNA, Transfer, Arg - chemistry; RNA, Transfer, Arg - metabolism; Transfer RNA; tRNA Arg; tRNA Asn; tRNA Asp; tRNA Tyr
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.140213797

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 tRNAsArgincluding 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 tRNAsArg, 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 tRNAArg-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAsArg, 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.