Supplementary Figure 5: Exu and Vts1p SAM domains use different surfaces for RNA binding.
From: The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease
a-b) Surface rendering of electrostatic charges of Exu SAM-like (a) and yeast Vts1p (b; PDB 2B6G, chain A) domain structure (see also Supplementary Fig. 2). a) Exu residues cross-linking with RNA (Fig. 5) are underlined. b) Vts1p residues shown to interact directly with the RNA (Aviv et al., NSMB 13, 168-176, 2006; Johnson and Donaldson, NSMB 13, 177-178, 2006; Oberstrass et al., NSMB 13, 160-167, 2006) are indicated. Structural alignment was done with cealign in Pymol (v 1.7.6.0). Scale of electrostatic charge distribution is the same for all domains (-5 to +5 kT/e).
c-d, f-g) A constant amount of 5'-fluorescein labelled oligo was incubated with increasing concentrations of recombinantly purified Exu wt or mutant. The fluorescence anisotropy data were fitted to the Hill equation to obtain the dissociation constant (Kd); mean Kd and standard deviation from three independent experiments are reported in tables (d, g). c, f) Data from a representative fluorescence anisotropy measurement, with the best fit plotted as a solid line. c, d) FA measurements of SAM-like domain mutants with (U)20. f, g) Affinity of Exu for oligo(U) RNAs of increasing length. e) Circular Dichroism (CD) spectra of Exu wt and the indicated mutants.
