Ogawa Tetsuhiro, Inoue Sakura, Yajima Shunsuke, Hidaka Makoto, Masaki Haruhiko
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Nucleic Acids Res. 2006;34(21):6065-73. doi: 10.1093/nar/gkl629. Epub 2006 Sep 8.
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.
大肠杆菌素E5是一种新型的大肠杆菌核糖核酸酶,它能特异性切割tRNA(Tyr)、tRNA(His)、tRNA(Asn)和tRNA(Asp)的反密码子。由于这种活性局限于其115个氨基酸长的C末端结构域(CRD),因此E5-CRD的识别机制备受关注。这四种tRNA底物在其反密码子环内共享独特序列UQU,并在Q(G的修饰碱基)和3' U之间被切割。与底物tRNA相对应的合成小螺旋RNA对E5-CRD完全敏感,并以与天然tRNA相同的方式被切割。E5-CRD的特异性决定因素是“反密码子” -1至+3位的YGUN。YGU是绝对必需的,小螺旋的敏感程度取决于N(反密码子的第三个字母),顺序为A > C > G > U,这与tRNA(Tyr)> tRNA(Asp)> tRNA(His)、tRNA(Asn)的敏感顺序一致。相比之下,我们表明GpUp是对E5-CRD严格保持特异性的最小底物。连续核苷酸的作用在环RNA和线性RNA之间不一致,这表明GpUp两侧的核苷酸延伸引入了结构限制,而这种限制通过形成特定的环结构(包括5'嘧啶和3' A)而降低。