Enzymatic footprinting of ribosomal complexes containing eIF1. cDNA products obtained by primer extension show protection of nucleotides 1811–1813 of 18S rRNA from RNase VI cleavage in the presence of eIF1. Reference lanes G, A, C, and T depict 18S rRNA sequence generated using the same primer.

Activities of eIF1 mutants in 48S complex formation. Toeprinting analysis of 48S complexes assembled on β-globin mRNA in the presence of 40S subunits, eIF2, eIF3, eIF4A, eIF4B, eIF4F, eIF1A, aminoacylated initiator tRNA, and the N-terminally truncated eIF1_Δ29 mutant (A) or Fe(II)-BABE-derivatized cysteineless and single-cysteine eIF1 mutants (B) as indicated. Reaction mixtures containing only β-globin mRNA are shown in lane 1 of each panel. Reaction mixtures corresponding to lane 2 of each panel contained all translation components except eIF1. Reference lanes A and G depict 18S rRNA sequence generated using the same primer. Complex I is arrested 21–24 nt from the 5′-end of β-globin mRNA. The 48S complex assembled on the AUG triplet is designated as “complex II (+15–17 nt from AUG).” The 48S complex assembled on the GUG triplet located 23 nt from the 5′-end of β-globin mRNA is designated as “+16 nt from GUG.”

Stereo view showing the position of eIF1 on the small ribosomal subunit. (A) Positions of directed hydroxyl radical cleavage in 18S rRNA and initiator tRNA (shown as colored spheres) mapped onto corresponding regions of 16S rRNA (gray) and tRNA (light brown) bound to the ribosomal P-site in a ribbon diagram of the crystal structure of the 30S ribosomal subunit from T. thermophilus (Yusupov et al. 2001). Colors of cleavage sites correspond to colors of cysteines on the surface of eIF1 in panel B and Figure 4A (C38 and C42, yellow; C57 and C61, green; C66, light green; C75, light blue; C91, magenta) from which hydroxyl radical cleavage occurs. The radius of the spheres is proportional to the efficiency of the cleavage: weak, medium, and strong. Ribosomal proteins are shown as light-blue ribbons. mRNA in the A- and P-sites is shown in red. (B) Close-up view of the region around the ribosomal P-site from panel A. The strongest cleavage sites in 18S rRNA modeled onto 16S rRNA structure are numbered and indicated by arrows. (C) Position of eIF1 (blue ribbon) on the 30S subunit from T. thermophilus (Yusupov et al. 2001) modeled using data from hydroxyl radical cleavage of 18S rRNA by Fe(II) tethered to different positions on the surface of eIF1. Colored spheres represent positions of cysteines on eIF1 from which hydroxyl radicals were able to cleave 18S rRNA. (D) Stereo view showing a model of the eIF1/30S/[P-site tRNA]/mRNA complex based on hydroxyl radical cleavage data and using the structure of the T. Thermophilus 30S subunit (Yusupov et al. 2001). eIF1 is in surface representation, painted by electrostatic potential (positive charge is blue and negative is red). The coloring of 16S rRNA, mRNA, tRNA, and ribosomal proteins in panels C and D is as in panels A and B.

Binding of eIF1 to the 40S ribosomal subunit. (A) Interaction of 40S subunits with T7-Tag antibody agarose-immobilized eIF1 in an in vitro binding assay. Ribosomal protein S6 was visualized by Western blotting. (B) Presence of eIF1 in ribosomal complexes isolated from sucrose density gradients. Prior to centrifugation, 40S subunits were incubated with eIF1 and different combinations of eIF3, eIF2–ternary complex, and poly(U) RNA as indicated. eIF1 in fractions containing 40S subunits was detected by Western blotting using anti-T7 tag antibodies.

Directed hydroxyl radical cleavage of 18S rRNA in 40S/eIF3/eIF1 complexes from Fe(II) tethered to different positions on the surface of eIF1. (A) Ribbon diagram of the structured domain of human eIF1. Spheres indicate positions of cysteines introduced on the surface of eIF1 for tethering of Fe(II)-BABE. The positions of cysteines from which hydroxyl radicals were able to cleave 18S rRNA are shown in colors (C38 and C42, yellow; C57 and C61, green; C66, light green; C75, light blue; C91, magenta). (B) Secondary structure of 18S rRNA with sites of directed hydroxyl radical cleavage shown as red bars. (C,E,G) Primer extension analysis of directed hydroxyl radical cleavage of 18S rRNA (in helices 24, 23, and 44, respectively) in 40S/eIF3/eIF1 complexes from Fe(II) tethered to positions on eIF1 as indicated. Reaction mixtures corresponding to the lanes marked “40S, eIF3” did not contain eIF1. Reaction mixtures corresponding to the lanes marked “Cys-less” contained the cysteineless eIF1 mutant. Reference lanes G, A, C, and T depict 18S rRNA sequence generated from the same primer. The positions of cleaved nucleotides are shown as black bars on the right. (D,F,H) Elements of helices 24, 23, and 44 of 18S rRNA with hydroxyl radical cleavage sites shown as red bars.

Directed hydroxyl radical cleavage of initiator tRNA in 43S complexes from Fe(II) tethered to different positions on the surface of eIF1. (A) Primer extension analysis of directed hydroxyl radical cleavage of initiator tRNA in 43S complexes that contained 40S subunits, eIF2, initiator tRNA, eIF3, and eIF1 from Fe(II) tethered to different positions on eIF1 as indicated. The reaction mixture corresponding to the lane marked “Cys-less” contained the cysteineless eIF1 mutant. The reaction mixture corresponding to the lane marked “no eIF1” did not contain eIF1. Reference lane G depicts tRNA sequence generated using the same primer. The positions of cleaved nucleotides are shown as black bars on the right. (B) Secondary structure of initiator tRNA with sites of directed hydroxyl radical cleavage shown as black bars.

Superposition of eIF1 and the C-terminal domain of IF3 (IF3-CTD). (A) Superposition of structural elements of eIF1 (blue ribbon) and the IF3-CTD (yellow ribbon; Biou et al. 1995), based on their positions on the small ribosomal subunit (Dallas and Noller 2001; this study). The orientation of eIF1 is as in Figure 6. (B) Comparison of the surface charge distribution of eIF1 and IF3-CTD based on their positions on the small ribosomal subunit (Dallas and Noller 2001; this study). Positively and negatively charged surfaces are blue and red, respectively. Both eIF1 and IF3-CTD are rotated 180° compared with panel A. The extensive rRNA-binding surface (front) and the tRNA contact surface (top) are indicated. The solvent-exposed surface facing the ribosomal E-site is on the left.