Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease

Abstract

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.

Figure 1

Minihelices used in this study. Each minihelix (MH) is referred to as XMH where X is the amino acid that is accepted by the parental tRNA. For example, YMH is the MH mimicking the anticodon arm of tRNA^Tyr. In the models, the top left is the 5′ end of each oligoribonucleotide, and each ribonucleotide is numbered from this position. GGXMH indicates a guanylyl-guanosine that was added at the 5′ end of XMH. If ribonucleotide X at position n was changed to Y, then it is indicated in parentheses as XnY. The horizontal lines indicate the putative H-bonds.

Figure 2

The potential of minihelices with wild-type or mutated sequences to be cleaved by E5-CRD. (A) YMH and KMH (5 µM each) were incubated with or without 10 nM of E5-CRD in 20 mM Tris–HCl (pH 7.5) and 20 mM KCl for 15 min at 37°C, and then analysed by electrophoresis on a 20% polyacrylamide gel containing 7 M urea (lanes 1, 2, 9 and 10, respectively). The visible mobility pattern was obtained with a FLA-3000 (FUJI FILM). Lane numbers are shown beneath the picture. The closed triangle indicates the band of intact MH. If cleavage of a MH occurred, a band of a shorter fragment appeared (indicated by the open triangle). Variants of YMH and KMH with a site-directed mutation were also incubated with E5-CRD and then analysed. Each mutation is indicated above the picture. The asterisk indicates the 5′-labelled G released from KMH and KMH(U8G) on cleavage between G1 and U2. (B) YMH, YMH(U7C) and KMH(U8G) were incubated with (lanes 3, 6 and 9, respectively) or without (lanes 1, 4 and 7, respectively) E5-CRD, and then analysed by electrophoresis. The bands were visualized with a FLA-3000 (FUJI FILM). Lane numbers are shown beneath the picture. In lanes 2, 5 and 8, fragments of partially digested YMH, YMH(U7C) and KMH(U8G) with 40 mM sodium carbonate, pH 9.0, respectively, were electrophoresed. The asterisk indicates the 5′-labelled G released from KMH(U8G) on cleavage between G1 and U2. (C) Graphical representation of the putative structure of 5′-labelled YMH and the cleavage site by E5-CRD. Cleavage (as indicated by a closed triangle) occurs between G8 and U9.

Figure 3

Time-dependent cleavage of various minihelices by E5-CRD. Various minihelices, summarized in Figure 1, were incubated and the extent of cleavage was examined at each time. (A) Minihelices GGYMH, GGHMH, GGNMH and GGDMH that mimicking tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp, respectively, were used. YMH was used as a control, and it was confirmed that 5′ extended two guanine residues do not affect the susceptibility. (B) A mutation was introduced at A10 of YMH, and the resulting YMH, YMH(A10G), YMH(A10U) and YMH(A10C) were used as substrates. (C) A mutation was introduced at U7 of YMH, and the resulting YMH, YMH(U7C), YMH(U7A) and YMH(U7G) were used as substrates. (D) YMH and its derivative, YMHL, with the stem structure broken, were used. Error bars indicate the standard deviation of three independent assays.

Figure 4

Cleavage of oligonucleotides by E5-CRD. (A) Various diribonucleotide diphosphates (125 µM) or those lacking 3′ phosphate (125 µM) were incubated with or without 50 nM of E5-CRD for 15 min at 37°C. In order to stop the reaction, 250 nM of ImmE5 was added. Then, the reaction solutions were directly applied on a reversed-phase column and the products were analysed. The upper (Control) and lower (E5-CRD added) charts of each panel were adjusted for retention times. (B) After the incubation of E5-CRD and GpUp, the reaction solution was mixed with authentic 3′-UMP, 3′-GMP or 2′,3′-cyclic GMP and applied on the column to compare retention times. 3′-UMP and 2′,3′-cyclic GMP were produced from GpUp by the cleavage reaction of E5-CRD.

Figure 5

pH dependence of the kinetic constants of E5-CRD with GpUp as the substrate. (A) k_cat values are plotted against pH. (B) K_m values are plotted against pH. (C) k_cat/K_m values are plotted against pH.

Figure 6

Schematic representation of the substrate recognition of E5-CRD. Amino acids forming hydrogen bonds are indicated in boldface. In the crystal structure, guanine (syn form) and uracil (anti form) bases are found to stack with the indole ring of Trp102 and the pseudo ring formed by Asp105 and Arg107, respectively.