Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease.
Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNA(Tyr), tRNA(His), tRNA(Asn) and tRNA(Asp). Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3' U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at -1 to +3 of the 'anticodon'. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNA(Tyr) > tRNA(Asp) > tRNA(His), tRNA(Asn). Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5' pyrimidine and 3' A.
Ogawa
Tetsuhiro
T
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Inoue
Sakura
S
Yajima
Shunsuke
S
Hidaka
Makoto
M
Masaki
Haruhiko
H
D000926:Anticodon / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D000926:Anticodon; D003087:Colicins; D015226:Dinucleoside Phosphates; D029968:Escherichia coli Proteins; D009711:Nucleotides; D009843:Oligoribonucleotides; C083084:guanylyl(3'-5')uridine 3'-monophosphate; D012343:RNA, Transfer; D004722:Endoribonucleases; C515828:colicin E5 protein, E coli