FIGURE 4.

Effects of substitution mutants in the CX₅CX₃H intervals of the TTP TZF domain on RNA binding. The binding of protein to RNA is expressed as the probe-bound fraction. Above the graphs are the amino acid sequences of the intervals under study. The underlined residues are those that were mutated and whose effects on binding are shown in the bar graphs. The results from mutants of finger 1 are shown as gray columns, and those of finger 2 are shown as white columns. A, substitution mutants in the CX₅C intervals. The results (mean ± S.D. (error bars)) are from four similar gel shift assays using 0.2 nm probe TARE5. B, binding assays with 0.2 nm TARE5 probe in serially diluted extracts, with total protein ranging from 10 to 1 μg. The probe-bound fractions (mean ± S.D.) are from four similar gel shift assays. C, superposition of the structural ensembles of finger 2 of WT TTP and mutant P157G (the RMSD is 10.37 Å; RMSD values were calculated from the superposition of the TZF domains of each mutant TTP with RNA-bound WT TTP) or finger 1 of WT TTP and mutant K123D (RMSD is 2.86 Å). The ribbon diagram of the WT TTP peptide backbone and side chains (sticks) are in yellow, the ribbon diagram of the mutant peptide backbone is in cyan, and side chains are shown in colors. RNA nucleosides are shown as gray spheres. Zinc atoms are shown as silver and copper (in P157G) or black and copper (in K123D) spheres. D, ribbon diagram of the peptide backbone of the human TTP TZF domain in complex with the ARE nonamer (sticks). The side chains of Glu¹⁰⁷ (orange spheres, at the lead-in to finger 1) and Arg¹⁶¹ (purple spheres, at the C+5 position of the finger 2 CX₅C region) are shown. Dashed arrows indicate nucleosides U5, U6, and A7 that interact with Glu¹⁰⁷ and Arg¹⁶¹. Zinc atoms (black spheres) and the zinc-coordinating residues (ball and stick) of each finger are also displayed. E, superposition of the peptide backbone of the TZF domains of WT TTP (yellow ribbon) in complex with the ARE nonamer (surface representation) and the mutant R161E (cyan ribbon; RMSD is 4.66 Å). Zinc atoms are shown as black and copper spheres. Residues Glu¹⁰⁷ and Arg¹⁶¹ (yellow mesh in the WT TZF domain) and Glu¹⁰⁷ and Glu¹⁶¹ (green mesh in the mutant R161E) are shown (left). The right panel depicts the detailed view of the interaction between Arg¹⁶¹ and Glu¹⁰⁷ (yellow sticks) in the WT and the non-interacting Glu¹⁰⁷ and Glu¹⁶¹ (color sticks) in the mutant R161E. RNA nucleosides U4, U5, U6, and A7 (gray spheres) are indicated. F, substitution mutants in the CX₃H intervals. The results (mean ± S.D.) are from four similar gel shift assays.