1. Volume 7, Issue 1, Pages 193-203 (January 2001)
A Conserved HEAT Domain within eIF4G Directs Assembly of the Translation Initiation Machinery 
Joseph Marcotrigiano, Ivan B. Lomakin, Nahum Sonenberg, Tatyana V. Pestova, Christopher U.T. Hellen, Stephen K. Burley 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1 Motifs Common to eIF4GI, eIF4GII, and eIF4G-Related Proteins
(Left) Schematic alignment of the amino acid sequences of various eIF4Gs and eIF4G-related proteins, highlighting conserved protein binding motifs. Sequence identities of the conserved eIF4A/IRES binding domains with eIF4GII are given as percentages. Binding sites for various proteins are color coded as follows: (PABP), blue; (eIF4E), orange; (eIF4A) central, red; (eIF4A) C-terminal, magenta; (Mnk-1), black. Blue arrow denotes picornaviral protease cleavage site.
(Right) Schematic diagram contrasting cap-dependent translation initiation from cap-independent, IRES-mediated translation initiation from a picornaviral IRES.
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Structural and Functional Characterization of the eIF4A/IRES Binding Domain of Human eIF4G
(A) Structure-based sequence alignments of the eIF4A/IRES binding domains of all known eIF4Gs and eIF4G-related proteins, including human eIF4GII (Gradi et al. 1998) (used in structure determination), human eIF4GI (Imataka et al. 1998), Saccharomyces cerevisiae TIF4631 and TIF4632 (Goyer et al. 1993), Triticum aestivum eIF-(iso)4G (Allen et al. 1992), human p97 (Imataka et al. 1997), and human Paip-1 (Craig et al. 1998). α-helical secondary structural elements are denoted with cylinders and labeled with repeat number and a or b. Dashed lines correspond to regions with poorly resolved electron density. Color scheme: red, identity; blue, high conservation. V8 protease cleavage sites are denoted by arrows. Location of eIF4G mutants: (1), M-1 (Imataka and Sonenberg 1997); (4), M-4 (Imataka and Sonenberg 1997); (7), mut(Ile749→Thr, Arg754→Ile) (Lomakin et al. 2000); (8), mut796-Ins8 (Lomakin et al. 2000); (R), RRM1 (Lomakin et al. 2000); (t), tif4632–6 (Neff and Sachs 1999); (A), reduced eIF4A binding, 756; (K), reduced IRES binding, 834; (B), reduced eIF4A and IRES binding, 814; (N), no effect, 798, 802, 803, 843, and 888. (See Table 2 for a complete description of eIF4G mutations.)
(B) Toeprint analyses of the interaction of eIF4GII (extended construct: 735–1097 and crystallization construct: 745–1003) with the EMCV IRES in the presence and absence of eIF4A. The full-length cDNA extension product is marked (e). A common stop site within the EMCV RNA used as an internal normalization standard is denoted by (N). The stop site due to binding of eIF4GII is indicated by (C786). Reference lanes (t), (c), (g), and (a) depict the EMCV IRES cDNA sequence.
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Structure of the eIF4A/IRES Binding Domain of eIF4GII
(A) Ribbon stereodrawing of the conserved central region of eIF4GII viewed along the cylindrical axes of the α helices, with the concave surface on the right and the convex surface on the left. α helices are labeled as in Figure 2A.
(B) Stereodrawing viewed perpendicular to the α helix axes, rotated 90° about the solenoid axis relative to (A), with the concave surface in the foreground. The intra- and interrepeat surfaces are located on the right and left, respectively.
(C) Stereodrawing viewed along the α-helix axes, rotated 180° about the solenoid axis relative to A, with the intrarepeat surface in the foreground and the concave and convex surfaces on the left and right, respectively.
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Comparison of the Middle Domain of eIF4GII with Other HEAT Repeat Proteins
Tubular α helix drawing comparing eIF4GII (745–1003) with the PR65/A subunit of PP2A (40–235) (Groves et al. 1999) and importin β (401–640) (Cingolani et al. 1999), using the view in Figure 3A (concave surface, right). Protein superpositions were determined by the DALI server (Holm and Sander 1993). For ease of comparison, α helices are numbered as in Figure 2A.
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 Site-Directed Mutagenesis of eIF4GII
(A and B) Toeprint analyses of the interaction of eIF4GII (735–1097) mutant polypeptides with the EMCV IRES alone (A) and with added eIF4A (B).
(C) Histogram showing the results of quantitative toeprinting of the EMCV RNA with eIF4GII. Solid (eIF4G) and open (eIF4G and eIF4A) bars represent average values obtained in five replicate experiments.
(D) Interaction of mutant eIF4GII (735–1097) polypeptides with immobilized eIF4A in a direct binding assay. eIF4A was visualized by Coomassie staining, and eIF4GII polypeptides were detected by Western blotting with anti-T7 tag antibodies.
(E) Primer extension analyses of 48S initiation complexes assembled on EMCV RNA using purified eIF1, eIF1A, eIF2, eIF3, eIF4A, eIF4B, methionyl initiator tRNA, and 40S ribosomal subunits with wild-type or mutant eIF4GII. The full-length cDNA extension product is marked (e), the position of the stop site due to binding of eIF4GII is denoted by (C786), and cDNA products labeled (AUG826) and (AUG834) terminated at stop sites 15–17 nt downstream of each initiation codon. Reference lanes (t), (c), (g), and (a) depict the EMCV IRES cDNA sequence.
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Surface Properties of eIF4A/IRES Binding Domain of eIF4GII
GRASP (Gilson et al. 1988) representation of the solvent-accessible surface of the eIF4GII calculated using a water probe radius of 1.4 Å.
(Left) The surface color-coded for calculated electrostatic potential: red < −9kBT and blue > 9kBT, where kB denotes Boltzmann constant and T, temperature.
(Right) The surface color-coded for the locations of mutants affecting binding to eIF4A (black) and EMCV IRES (cyan). The disordered 2b-3a loop (residues 823–831) is represented by closed dots. (A), (B), and (C) views are identical to those shown in Figures 3A, 3B, 3C, respectively.
Molecular Cell 2001 7, DOI: ( /S (01) )