Figure 3.

Directed hydroxyl radical cleavage of Met-[³²P]tRNA_i^Met by Fe(II)-BABE-derivatized eIF2 in 48S complexes.

(a) eIF2ΔC-γR118C-Fe(II)-BABE cleavages. Hydroxyl radical cleavage products from 48S complexes were resolved on 10% (w/v) denatured polyacrylamide gels, and cleavage sites on [³²P]tRNA_i^Met were determined by comparison with samples containing eIF2ΔC (WT) (lane 2). Sites of enhanced cleavage with eIF2ΔC-R118C are boxed. The tRNA ladders were prepared by digesting Met-[³²P]tRNA_i^Met with RNase T1 (cleaves 3′ of G residue, lane 3) or by base cleavage (lane 4). The tRNA residue numbers are shown at the left.

(b) Sites (colored magenta) of eIF2ΔC-γR118C-Fe(II)-BABE directed hydroxyl radical cleavage on Met-[³²P]tRNA_i^Met are shown on the secondary (left) and three-dimensional (pdb code: 1YFG¹⁹, middle and right) structures of tRNA_i^Met.

(c) Met-[³²P]tRNA_i^Met cleavages in 48S complexes assembled with eIF2ΔC-γK507C-Fe(II)-BABE and eIF2ΔC-βS264C-Fe(II)-BABE, as in panel a. Cleavage products were resolved on 10% (left) or 20% (right) (w/v) denatured polyacrylamide gels.

(d,e) Summary of Met-[³²P]tRNA_i^Met cleavages in panel c, depicted as in panel b.

(f) aIF2γ-GDPNP-tRNA_i^Met TC model showing the sites of tRNA_i^Met (cyan) cleavages by hydroxyl radicals generated at residues K507 (blue) and R118 (magenta). The model was generated by docking the structure of yeast tRNA_i^Met (pdb code: 1YFG¹⁹) on the aIF2γ TC structure from Fig. 1b. The RIT1 catalyzed 2′-O-ribosyl phosphate modification at residue 64 of tRNA_i^Met is shown as orange spheres.