Volume 5, Issue 1, Pages 109-119 (January 2000) The eIF1A Solution Structure Reveals a Large RNA-Binding Surface Important for Scanning Function  John L. Battiste, Tatyana V. Pestova, Christopher U.T. Hellen, Gerhard Wagner  Molecular Cell  Volume 5, Issue 1, Pages 109-119 (January 2000) DOI: 10.1016/S1097-2765(00)80407-4

Figure 1 Sequence Homology of eIF1A-like Proteins across All Kingdoms Sequence alignments adapted from the paper by Kyrpides and Woese 1998. Amino acids that have greater than 50% identity over 12 sequences analyzed by Kyrpides (more than shown in figure) are shaded. Identities between human and yeast are shown with black circles (65% identity). Side bars indicate sequences from eukaryotes (E), archaebacteria (A), and eubacteria (B/IF1). The secondary structure of human eIF1A (determined in this paper) over the region that has observable long-range NOEs is shown below the sequence (wavy lines indicate coils or turns; arrows indicate strands; cylinders indicate helices). The secondary structure elements are labeled β for β strand, α for α and 310 helices, L for loops connecting β strands or helices, N for amino-terminal strand, C for carboxy-terminal strand. Loop numbering indicates the secondary structure elements connect by the loops (adapted from Murzin 1993). For instance, L12 connects strand 1 and 2, and L3α connects strand 3 and the subsequent helix 1. Organisms are: HUMAN, Homo sapiens; YEAST, Saccharomyces cerevisiae; METJA, Methanococcus jannaschii; ARCFU, Archaeoglobus fulgidus; ECOLI, Escherichia coli; BACSU, Bacillus subtilis. Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)

Figure 2 Structure of eIF1A (A) Stereo view of the superposition of 20 calculated structures from residues 25–117. Backbone atoms are shown in blue, green, and black for β strands, helices, and loops, respectively. Side chain heavy atoms for well-defined residues in the core of the protein are shown in red. Plot produced with the program MOLMOL (Koradi et al. 1996). (B) Ribbon diagram of the core of eIF1A (residues 25–117). The OB and helical domains are colored gray and red, respectively. Secondary structure elements defined in Figure 1 are labeled. Diagram produced with the program Molscript (Kraulis 1991). Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)

Figure 3 Binding of eIF1A to Single-Stranded RNA (A) Overlaid 15N-HSQCs at 750 MHz of eIF1A before and after titration with RNA (25°C). Black contours are free eIF1A (0.11 mM and 200 mM NaCl), and red contours are after addition of 0.33 mM RNA. Backbone amides that shift more than 18 Hz in either dimension are labeled. The NH of the tryptophan ring is labeled εNH. (B) Titration calorimetry curve. Plot is total RNA concentration (μM) versus total heat liberated (μcal). Solid line is a fit of the data to a simple bimolecular association (1:1). The buffer for the calorimetry experiment contains lower NaCl concentrations (100 mM) than the NMR experiments. Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)

Figure 4 Mapping of Chemical Shift Changes onto the Structure of eIF1A (A) Bar graph of the absolute value of the chemical shift change upon addition of 0.44 mM RNA Δσ = ∣σfree − σbound∣. Values for two different RNAs are plotted: up is GCCACAAUGGCA, while down is GGACUUCG. The y axis is plotted as a shift index equal to ΔσH + ΔσN/5 (in ppm) to compensate for the larger chemical shift range in 15N. The 15N contribution to the index is shown in black with the 1H contribution in red. (B) Backbone amides that shift more than 18 Hz in either dimension are colored red in the ribbon diagram of eIF1A (Molscript [Kraulis 1991]). These are backbone amides for 40–42, 44–46, 59–62, 64–65, 67, 70, 81, 83, 86, 88, 92, 95, 96, 99, 100, 108, and 111–117 and the side chain εNH for W69 (66 Hz). Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)

Figure 5 Molecular Surfaces of eIF1A (A) Three views of the electrostatic surface of eIF1A produced with the program GRASP (Nicholls et al. 1991). Positive charge is colored blue and negative red (±40 kT). Surfaces to the right are progressively rotated 90° counterclockwise around the vertical axis of the paper. eIF1A is in a different orientation from the other figures with the far right panel looking down the axis/hole of the β barrel (approximately 90° clockwise rotation of Figure 4B around horizontal axis of paper). Secondary structure elements are labeled for orientation. (B) Surface of amino acids that have backbone amide chemical shift changes upon binding RNA are colored green (same residues as Figure 4B). Yellow surfaces are amino acids that have undetectable amide resonances due to intermediate conformational exchange in the unbound protein and are not available as probes of RNA binding. The three surfaces have the same orientation as in (A). Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)

Figure 6 The Influence of Substitutions in the RNA-Binding Surface of eIF1A on Function (A, B, and C) Toeprint analysis of ribosomal complexes formed in assembly reactions that contained (A and B) β-globin mRNA or (C) EMCV RNA and eIF2, eIF3, eIF4A, eIF4B, eIF4F, initiator Met–tRNA, GTP, ATP, 40S ribosomal subunits, wild-type or mutant eIF1A (as indicated), and eIF1 ([A], lanes 3–11; [B], lanes 2–7; [C], lanes 2–10). Wild-type or W69A eIF1A and 8 mM Mg2+ were added to reactions at the indicated times in (B). The positions of ribosomal complexes on these mRNAs were determined by primer extension inhibition analysis, using reverse transcriptase to extend primers annealed to a single position on each mRNA. Full-length cDNA is marked E. Other cDNA products terminated at the sites indicated on the right. Reference lanes C, T, A, and G depict β-globin or EMCV sequences, as appropriate. (D) The effect of wild-type and mutant eIF1A proteins on 43S preinitiation complex formation. Reaction mixtures containing 40S ribosomal subunits, [35S]Met–tRNA and wild-type or mutant eIF1A (as indicated) were incubated at 37°C for 5 min (see the Experimental Procedures). The formation of 43S preinitiation complexes was assayed by centrifugation on 10%–30% sucrose density gradients. The position of 43S complexes is indicated. Fractions from the upper part of the sucrose gradient have been omitted for clarity. Molecular Cell 2000 5, 109-119DOI: (10.1016/S1097-2765(00)80407-4)