Interaction of Translation Initiation Factor 3 with the 30S Ribosomal Subunit  Anne Dallas, Harry F Noller  Molecular Cell  Volume 8, Issue 4, Pages 855-864 (October 2001) DOI: 10.1016/S1097-2765(01)00356-2

Figure 1 Chemical Footprinting of Fe(II)-Derivatized IF3 Variants on 16S rRNA Primer extension showing the kethoxal footprint of IF3-HisTag (wild-type) and Fe(II)-BABE-derivatized IF3 variants at positions G700 and G703 on 16S rRNA in the 30S subunit. In both panels, A and G are sequencing lanes. Lanes labeled K and 30S are unmodified 30S subunits and kethoxal-modified 30S subunits, respectively. IF3 is kethoxal-modified 30S-IF3-His6 complex. In the left panel, –cys is kethoxal-modified cysteine-free IF3-30S, and all other lanes are kethoxal-modified N domain Fe(II)-IF3-30S complexes, as indicated at the top of each lane. In the right panel, all other lanes are C domain Fe(II)-IF3-30S complex treated with kethoxal, as indicated Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 2 Hydroxyl Radical Footprinting of IF3 on 16S rRNA (A) Primer extension analysis of the hydroxyl radical footprint of IF3 on 16S rRNA in the 30S subunit. Lanes from left to right are as follows: A and G, sequencing lanes; K, unmodified 30S subunit; 30S, 30S subunits exposed to hydroxyl radicals. Subsequent lanes are initiation factor-30S complexes (as labeled) exposed to hydroxyl radicals. The bars at the right of each autoradiogram indicate regions of protection. (B) IF3-dependent protection of 16S rRNA in 30S subunits from free hydroxyl radicals mapped onto the secondary structure of 16S rRNA. Dot sizes indicate the extent of protection Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 3 Directed Hydroxyl Radical Probing of 16S rRNA from Different Positions on the Surface of IF3 (A) Ribbon diagrams of the crystal structures of the N and C domains of IF3 from Bacillus stearothermophilus (Biou et al., 1995). Spheres indicate the Cα positions of engineered cysteine residues used to tether Fe(II), numbered according to the corresponding residue in E. coli. (B) Directed hydroxyl radical cleavage of 16S rRNA in 30S subunits from Fe(II)-IF3 detected by primer extension analysis. A and G are sequencing lanes. All other lanes are 30S-IF3 complexes that were probed with Fe(II) tethered to a different IF3 position, as indicated, including a cysteine-free control reaction (−cys). Labels at the left of each autoradiogram indicate the sequence of 16S rRNA. Bars at the right of each panel indicate regions of cleavage by hydroxyl radicals. (C) A summary of the location of hydroxyl radical cleavages in the central, the 3′ major, and the 3′ minor domains of 16S rRNA (shaded gray, clockwise from left) from Fe(II)-IF3 bound to 30S subunits. Cleavage strengths, assigned as strong, medium, or weak, are proportional to the size of the filled circles Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 4 Directed Hydroxyl Radical Probing of Initiator tRNA from Different Positions on IF3 (A) Autoradiograph of 5′ end-labeled tRNAMetf showing cleavage by hydroxyl radicals generated from Fe(II)-IF3. Lanes are labeled according to the site of attachment of Fe(II)-BABE to IF3. Cleavages are indicated by bars at the right side of the gel Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 5 Positioning IF3 on the 30S Subunit (A) Hydroxyl radical footprint of IF3 mapped onto a ribbon diagram of the crystal structure of the 30S subunit from Thermus thermophilus. The strongest protections are colored magenta, and weaker protections are colored a lighter pink. Base-specific protections are represented as red spheres. (B) A ribbon diagram of IF3 (yellow) docked onto the 30S subunit footprint. The N and C domains are labeled N and C, respectively. (C) Model of the interaction of IF3 (black ribbon) with the 30S subunit as determined by directed hydroxyl radical probing and hydroxyl radical footprinting. Nucleotides cleaved by Fe(II)-IF3 are mapped onto a ribbon diagram of 16S rRNA in the 30S subunit from the crystal structure of the Tth ribosome (Yusupov et al., 2001). Ribosomal proteins S7 and S11 are colored green, and the 16S rRNA backbone is traced in white, except where it is cleaved by Fe(II)-derivatized IF3. Nucleotides cleaved from probing positions 97 and 135 are blue (strong hits) and lighter blue (weaker hits), while nucleotides cleaved from the N domain probes are colored red (strong hits) and lighter red (weak hits). Cleavages from position 104 are shaded gold. The corresponding probing positions are represented as spheres and are colored to match their respective cleavage targets Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 6 The Position of IF3 Relative to Initiator tRNA, mRNA, and IF1 (A) Views of the IF3-30S model with initiator tRNA bound to the P site and the location of IF1 as determined by the crystal structure (Carter et al., 2001). 16S rRNA and small subunit proteins are shaded light and dark gray, respectively. IF3 is represented in CPK and is colored red. IF1 is shaded blue. Initiator tRNA is traced in yellow, and mRNA is colored purple. (B) A closeup view of IF3 and P site-bound initiator tRNA showing the cleavages from directed probing experiments. Initiator tRNA is colored yellow, except where cleaved by Fe-C135(green) and Fe-C76 and Fe-C80 (blue). The corresponding probing positions are colored similarly on IF3 (gray). mRNA is represented in purple Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)

Figure 7 The IF3 C Domain Occupies the Position of Helix 69 of 23S rRNA (A) A view of the interaction of helix 69 (yellow) of 23S rRNA with helices 23, 24, and 45 of 16S rRNA (blue). The sites of contact between 23S rRNA and 16S rRNA are colored purple. (B) A view showing the overlapping binding site on the 30S subunit of the C domain of IF3 (red) with helix 69 of 23S rRNA (yellow) Molecular Cell 2001 8, 855-864DOI: (10.1016/S1097-2765(01)00356-2)