Structure–activity relationships in Kluyveromyces lactis γ-toxin, a eukaryal tRNA anticodon nuclease

Requirements for anticodon nuclease activity in vitro. (A) Nuclease reaction mixtures (10 μL) containing 20 mM Tris-HCl (pH 7.5), 2 M TMAO, and 200 fmol 5′ ³²P-labeled 17-mer RNA (depicted in D) were supplemented with either 1 mM DTT, 10 mM MgCl₂, 10 mM EDTA, or 5 pmol γ-toxin where indicated by “+” and then incubated at 4°C for 30 min. The products were analyzed by denaturing PAGE and visualized by autoradiography. The extents of RNA cleavage are indicated below the lanes. (B) Reaction mixtures (10 μL) containing 20 mM Tris-HCl (pH 7.5), 200 fmol 5′ ³²P-labeled 17-mer RNA, 50 fmol γ-toxin, and TMAO as specified were incubated at 4°C for 30 min. The products were analyzed by denaturing PAGE in parallel with a size marker ladder generated by partial alkaline hydrolysis of the 5′ ³²P-labeled 17-mer RNA (lane OH⁻). An autoradiogram of the gel is shown. (C) Reaction mixtures (10 μL) containing 20 mM Tris-HCl (pH 7.5), 2 M TMAO, 200 fmol 5′ ³²P-labeled 17-mer RNA with a ribose at the wobble nucleoside (2′-OH) (left), with a deoxyribose at the wobble nucleoside (2′-H) (middle), or with a 2′-fluoro sugar at the wobble nucleoside (2′-F) (right), and 100 fmol γ-toxin (lanes +) were incubated at 4°C for 30 min. Enzyme was omitted from control reactions in lanes –. The reaction products were analyzed by denaturing PAGE in parallel with size marker ladders generated by partial alkaline hydrolysis of the respective 5′ ³²P-labeled 17-mer RNAs (lanes OH⁻). An autoradiogram of the gel is shown. (D) The 17-mer RNA oligonucleotide substrate corresponding to the anticodon stem–loop of tRNA^Glu is shown. The site of cleavage by γ-toxin 3′ of the wobble base of the tRNA^Glu anticodon nucleoside is indicated by the arrow.