Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease
2006
Ogawa, Tetsuhiro | Inoue, Sakura | Yajima, Shunsuke | Hidaka, Makoto | Masaki, Haruhiko
Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNATyr, tRNAHis, tRNAAsn and tRNAAsp. 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 tRNATyr > tRNAAsp > tRNAHis, tRNAAsn. 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.
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