Figure 3.

Structure and RNA-binding of ZnK1-5 Air2p. (A) The lowest energy structure of the Air2p ZnK1-5. The protein is shown as a ribbon model (in red) with the zinc-coordinating residues (CCHC) shown in yellow. The zinc ions are shown in blue. (B) Close-up view of the ¹H-¹⁵N TROSY spectrum, showing the N-H correlations of histidines that are involved in the coordination of zinc ions. (C) Air2p ZnK1-5 binds A15 RNA. Binding isotherms for equilibrium binding of ZnK1-5 Air2p to fluorescently labeled A₁₅ RNA, monitored by fluorescence anisotropy. (D) Air2p ZnK1-5 binding to dA₁₅ DNA. The ionic strength and pH of the binding buffer was the same for both measurements. The dissociation constant (K_d) was calculated from the best fit of data using a single-site binding isotherm. (E) ¹H-¹⁵N TROSY spectra of Air2p ZnK1-5 alone (in blue) and in the presence of one equivalent of hypomodified tRNA_i^Met (in red; ¹H-¹⁵N HSQC) at 20°C. (F) Summary of chemical shift perturbations and line broadening of Air2p ZnK1-5 upon binding to hypomodified tRNA_i^Met. Affected residues (qualitatively described by perturbation index; yes=1, no=0) are plotted against the amino-acid residue number. The assignments of residues indicated by asterisks could not be obtained. (G) Solvent-accessible surface representation of the RNA-binding ZnK of Air2p (ZnK2, ZnK3 and ZnK4) colored by electrostatic potential (blue, positive; red, negative) of the representative structure.