Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5. but weakly to the surface of whereas 3c1 and 3c2 a stoichiometric complex with 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.


Correspondence kasano@ksu.edu
In Brief During translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome. Obayashi et al. propose that the N-terminal domain of eIF3c reaches into the decoding center to not only anchor the gate-keeper eIF1 but also facilitate eIF1 release on AUG selection. eIF5 appears to play a role in this regulation.
The high accuracy of initiation in eukaryotes results from suppressing initiation from non-AUG codons like GUG and UUG. This stringency is imposed partly by eukaryotic initiation factors (eIFs) that bind the small (40S) ribosomal subunit in the 43S preinitiation complex (PIC), i.e., eIF1A, eIF1, eIF2, eIF3, and eIF5 (Asano, 2014;Hinnebusch, 2014). Like its bacterial counterpart IF1, eIF1A binds the 40S A-site. The other four factors engage in numerous mutual interactions to form the multifactor complex (MFC) with Met-tRNA i Met bound to eIF2-GTP in the ternary complex, whereby MFC can be isolated free of ribosomes from various eukaryotes ( Genetic studies have revealed that eIF3c-NTD contains two distinct elements with opposing roles in initiation accuracy. Box12 is required for accurate initiation, and substitution mutations in this element increase non-AUG initiation (for the Suior suppressor of initiation codon mutation phenotype). The Box6 element is required for initiation at non-AUG codons, and substitutions in Box6 suppress effects conferred by a Suimutation (for the Ssuor suppressor of Sui phenotype) (Kará sková et al., 2012; Valá sek et al., 2004). Henceforth, Box6 and Box12 are designated as an Ssu + (Box6 Ssu+ ) and a Sui + element (Box12 Sui+ ), respectively. Certain Box6 or Box12 mutations decrease eIF1 binding to the eIF3c-NTD, suggesting that the eIF3c-NTD helps to stabilize eIF1 in the PIC not only during mRNA scanning, but also during the switch to the closed state upon start codon selection. Herein, we employed a battery of biophysical methods including nuclear magnetic resonance (NMR) spectroscopy to dissect eIF3c-NTD into three units, 3c0, 3c1, and 3c2 and locate the latter two within the recently solved cryo-EM PIC structure (Erzberger et al., 2014). Based on physical interaction studies involving eIF1, eIF3c-NTD, and eIF5, we propose that, by interacting with the N-terminal unit 3c0, eIF5 modulates the ability of eIF3c-NTD to either anchor or release eIF1. Our model explains distinct contributions of eIF3c Box6 Ssu+ and Box12 Sui+ to the accuracy of start codon selection in vivo.
Based on these results, we identify 3c1 as the core eIF1-binding site in eIF3c-NTD. Low-affinity eIF1 binding by flanking region 3c0 containing Box6 Ssu+ contributes to the high-affinity eIF1 binding (1 mM) by fragments containing 3c1 and 3c0, likely through interaction with more than one site on eIF1. Because we failed to generate an eIF3c segment containing only 3c1, the contribution of C-terminal flanking 3c2 remained unclear. However, based on the low-affinity eIF1 binding to eIF3c-E 87-163 , 3c2 containing Box12 Sui+ likely contributes to the relatively high-affinity binding (8 mM) observed for eIF3c-D 58-163 . CSP Mapping with 15 N-eIF3c-NTD Identifies aa Involved in eIF1 Binding Next, we used NMR chemical shift perturbation (CSP) mapping to delineate eIF3c residues directly involved in eIF1 binding. We first determined the structure of eIF3c-NTD by NMR spectroscopy using [ 13 C, 15 N] eIF3c-B 36-163 segment (see Supplemental Information and Table S3 for details), which demonstrated that the region covering most of 3c2 (residues 105-159) folds into a-helical globule ( Figure 1C). The eIF3c backbone resonance assignments were then used for CSP studies. As shown in Figure S3, CSPs induced by eIF1 binding are nearly identical between 15 N-eIF3c-A 1-163 and -B 36-163 , which is consistent with our GST-pull down and ITC studies (Figures 1A, S1B, S1C, and S2) (Kará sková et al., 2012). We further observed large CSPs for A67 (circled in blue in Figure 1D; indicated by arrow in Figure S3, lower panels), and E51 residues (circled in blue in Figure 1D) accompanied by the strong resonance line broadening in the stretch: K 68 (P)YG(P)DWFKK 77 (K68, Y70, and F75 highlighted in Figure 1D; others highlighted in Figures S3A and S3B; prolines are in parentheses). In contrast, all CSPs in 3c2 were minor (<0.04 ppm) ( Figure 1E) except for F90, which was considered spurious inasmuch as it was not eliminated by an eIF1 mutation that abolishes interaction with eIF3c (shown below in Figure S4, panel 1). Collectively, these results indicate that the eIF1-binding site on eIF3c NTD resides in the area covering Box6 Ssu+ of 3c0 (containing E51) and core region 3c1 (aa 58-87, contains A 67 K(P)YG(P)DWFKK 77 ) ( Figures 1A and S1A).
Structure of eIF3c-NTD 105-159 and Integrated Modeling of eIF3:eIF1:40S Complex Structure Define Two Globular Units within eIF3c-NTD In a recent cryo-EM study of the eIF1/eIF3/40S complex, which integrated extensive crosslinking information, it was proposed that the eIF3c-NTD projects from the solvent side along the 40S subunit into the decoding center, where eIF1 is bound (Erzberger et al., 2014). However, structural information for the eIF3c-NTD was lacking. We therefore incorporated NMR structure of eIF3c segment 105-159 ( Figure 1C) into the integrated modeling platform and calculated a new localization for the whole eIF3 complex ( Figure S5). The resulting localization densities for eIF3c-NTD had a resolution of 18 Å (Figure 2A, left), guided by four high-confidence crosslinks (Figure 2A, right), which is a clear improvement from the 38 Å precision in our previous model (Erzberger et al., 2014). The eIF3c-NTD is resolved into two globular units that span the 60 Å distance between eIF1 and rpS13/uS15 (Figures 2A and S5). The one is located near rpS13/uS15 and was assigned as a-helical globular structure in 3c2 (aa 105-159) shown in Figure 1C. The other is adjacent to eIF1 and, thus, was assigned as the core eIF1-binding region 3c1 (aa 59-87) ( Figure 1A). Indeed, recent medium-resolution (A) Location of eIF1-binding site in eIF3c primary structure (orange rectangle), highlighting regions of Ssu and Sui mutation sites, Box6 and Box12 (boxes with numbers). Orange schematics below indicate functional elements identified in eIF3c-NTD, 3c0, 3c1, and 3c2. The lines further beneath depict eIF3c deletion constructs used in this study. Dotted lines define the boundaries of eIF3c-NTD regions i-iv ( Figure S1A). Table summarizes the results of ITC analysis for eIF1 binding, K d and N (stoichiometry; number of eIF3c molecules bound to a eIF1 molecule) (see Figure S2). Weak, the weakest binding observed with eIF3c-G in GST pull-down. (B) GST pull-down assay. Approximately 5 mg of indicated GST-eIF3c fusion proteins (0.15-0.2 nmol) was allowed to bind 70 mg of recombinant eIF1 (5 nmol; 25 mM) in E. coli lysates (lanes labeled ''+'') and the protein complexes pulled down and analyzed with 5% input amounts of lysates by SDS-PAGE, followed by immunoblotting with anti-yeast eIF1 (bottom) and Ponceau staining (top). -, uninduced E. coli lysates were used as a negative control. In lanes 7 (*) and 10 (**), 10% and 90% of the GST-eIF3c-F complex were analyzed, respectively.  Figure 1A) was not localized in the eIF3:eIF1:40S structure, presumably because it cannot bind eIF1 when eIF1 is bound to the 40S subunit (as discussed below). Therefore, integrative modeling that incorporates the NMR structure of eIF3c 105-159 pinpointed the locations of the 3c1 and 3c2 elements within the PIC, with 3c1 directly contacting eIF1.
NMR Evidence that eIF3c-NTD Segments 3c1-3c2 Interact with a Limited Surface of eIF1 Compatible with 40S Binding eIF1 comprises an unstructured N-terminal tail (NTT) and a globular domain with a b1-b2-a1-b3-b4-a2-b5 fold (Fletcher et al.,  Table S4). To determine the eIF1 residues contacted by the 3c1-3c2 units in the complex formed with eIF3c D 58-163 , we performed CSP experiments using 15 N-eIF1. As shown in Figure 3B and summarized in Figure 3A, strong CSPs were observed for R53, K56, I93, and L96 residues on eIF1 thereby indicating that these residues on eIF1 direct its interaction with eIF3c D 58-163 . In contrast, resonances corresponding to residues within or nearby the two eIF1 ribosome-binding sites (Martin-Marcos et al., 2013; Rabl et al., 2011), including K60 at the a1 C terminus and T40/T41 near the b1-b2 loop (loop 1), were only marginally affected ( Figures  3A and 3B). As summarized in Figure 4A, the eIF3c-D 58-163 -binding site on eIF1 comprises the N-terminal and central portions of a1 and the adjacent hydrophobic area containing I93 (residues  painted red or orange). In agreement with this, the previous EM study showed that K56 on eIF1 crosslinks with K92 on eIF3c, which is located in the vicinity of 3c1 region ( Figure 2A). Because eIF1 interacts with the ribosome via residues K59 and K60 at the C terminus of a1, and R36 in loop 1 (residues painted cyan in Figure 4A), stoichiometric eIF1 binding to the 3c1-3c2 segment of eIF3c appears to be compatible with eIF1:ribosome association.
In conclusion, eIF3c-D 58-163 containing 3c1 and 3c2, but not 3c2 alone binds eIF1 in a manner compatible with eIF1 binding to the ribosome. Thus, the role of 3c2 in stimulating eIF1 binding to eIF3c-NTD, if any, appears to be indirect.

NMR Evidence that Segment 3c0-Box6 Ssu+ Interacts with the Ribosome-Binding Surface of eIF1
Relative to 3c1-3c2 segment D 58-163 , fragment C 36-87 , containing 3c1 and part of 3c0, displayed CSPs of greater intensity for a larger number of 15 N-eIF1 resonance peaks ( Figure 3D). Herein, in addition to R53 and L96 eIF1 residues, extensive CSPs were also observed for D61, A63, and N65, which are localized in the a1-b3 loop, T41 in b2 near loop 1, and L80 (Figure 3D, summarized in Figure 3A). This suggests that C 36-87 fragment binds an entire side of b sheets 1-4 of eIF1 that is adjacent to K60 at the a1 C terminus, the 40S contact site, and is likely to overlap with the second 40S contact site in loop 1, R36 (cyan lettering in Figure 4B). Interestingly, the resonance corresponding to I93 was slightly shifted in the presence of C 36-87 without attenuation of its signal ( Figure 3D, yellow for weak/moderate interaction) but did not disappear (line broadening) as observed for D 58-163 (red in Figure 3B). As summarized in Figure 4B, this pattern suggests that C 36-87 still retains interaction with R53 and L96 of eIF1 through the core element, 3c1, while its interaction with eIF1-I93 is diminished due to lack of 3c2. This supports an indirect stimulatory role for 3c2 in eIF1 binding to eIF3c-NTD (dotted line in Figure 4A).
Importantly, these data also suggest that the presence of the C-terminal half of 3c0 in C 36-87 confers more extensive interactions with the ribosome-binding surfaces of eIF1 ( Figure 4B). eIF1 residues assigned to the resonances are shown with their aa numbers. aa of high relevance (R53, K56, K60, I93, L96) are highlighted in red. Bottom, the eIF1 residues affected by each eIF3c segment are painted orange or yellow for strong or moderate CSP of >0.1 ppm or 0.050.1 ppm, respectively, in the ribbon diagram of yeast eIF1 structure. The eIF1 residues whose resonances caused line broadening were painted red. Locations of aa of high relevance are indicated. Prolines (11,46, and 72) and unassigned residues (23, 34-36, 66, and 107) are painted green and gray, respectively. In (B), note that, upon eIF3c-D 58-163 addition, the cross peak for K60 was shifted only slightly (green arrowhead in the spectrum), overlapping with that for K56, which shifted a greater distance (long black arrow). See also Tables S1 and S4. This is in agreement with its N value in ITC experiments of 0.7, indicating more than one binding site on eIF1 ( Figures 1A and  S2B). Hence, we propose that 3c0 does not engage eIF1 in the scanning PIC because its binding site on eIF1 overlaps with the 40S-binding surface.
The conclusion that the C-terminal half of 3c0 containing Box6 Ssu+ engages ribosome-binding surface of eIF1 is further supported by ITC analysis indicating that the K37E substitution in eIF1 loop 1 reduces eIF1 binding to eIF3c-NTD by 4-fold ( Figures 5B  and S6A). Importantly, 3c0:eIF1 interaction may explain the Ssuphenotype of the Box6R mutation (Valá sek et al., 2004). Notwithstanding that due to the relatively low affinity of eIF1 for eIF3c-NTD (K d = 1 mM) 3c0 is unlikely to displace eIF1 from PIC (eIF1:40S subunit K d = 1-10 nM) (Martin-Marcos et al., 2013), by competing with the eIF1:40S subunit interaction 3c0 may increase the chance that eIF1 is inappropriately released from the 40S sub-  Table S1. unit at a non-AUG codon. By disrupting this competition, the Box6R mutation of 3c0 is expected to stabilize the scanning PIC and diminish non-AUG initiation (Ssuphenotype). Thus, combined with the genetic findings (Valá sek et al., 2004), the CSP study in Figure 3D suggests that 3c0:eIF1 interaction impedes eIF1 binding to the ribosome.
Arg-53 and Leu-96 of eIF1 Make Critical Connections to the eIF3c-NTD within the Scanning PIC CSP analysis implicated eIF1 residues R53 and L96, in the N-terminal end of a1 and nearby hydrophobic patch, in interaction with all three eIF3c-NTD constructs that bind eIF1 with strong affinity ( Figures 3B, 3D, and 3E). Accordingly, we tested the effect of substituting these residues on eIF1 binding to eIF3c-B 36-163 in vitro. As controls, we examined eIF1 substitutions K56A and K60E, which are involved in 40S binding (see Figures 3A and 5A for eIF1 residues altered). In the ITC assay, R53S substitution reduced eIF3c binding below the detection limit, whereas L96P substitution reduced the affinity by 10-fold ( Figures 5B and  5C). In contrast K56A and K60E exerted little effect on eIF3c-NTD:eIF1 binding ( Figures 5B and S6A), which is consistent with NMR data. These results were verified by CSP experiments (Figures 5F and S4). These results also agree with our previous GST pull-down assays indicating that simultaneous substitution of eIF1 residues K52, R53, K56, K59, and K60 distributed along a1 (sui1-M5) (Reibarkh et al., 2008) and I93, L96, and G97 in the hydrophobic patch (sui1-93-97) (Cheung et al., 2007) reduces eIF1 binding to eIF3c (italicized are aa whose single substitution was found here to reduce the interaction).
To test this tenet, we examined the effects of L96P on eIF1 interactions with its other known binding partners: the 40S subunit, the eIF2b-NTT, and the eIF5-CTD ( Figure 5A). We determined the K d for the 40S$eIF1 complex by measuring changes in fluores-  Figure 5A for summary of interaction involving eIF1-L96). Thus, the strong Suiphenotype of L96P ( Figure 6A) likely arises from combined defects of reduced eIF1 binding to the eIF3c-NTD, eIF5-CTD ( Figure 5E), and perhaps the 40S subunit ( Figure 5B).
Despite the fact that eIF1 substitution R53S essentially abolishes binding to the eIF3c-NTD ( Figure 5B), it has no effect on initiation accuracy ( Figure 6A), implying that eIF1-R53S retains other interactions with the PIC that compensate for impaired interaction with eIF3c. Employing a variation of the FA assay in which excess unlabeled eIF1 competes with wild-type (WT)labeled eIF1 for ribosome binding ( Figure 5D, left), we found that R53S has only a slight effect on 40S binding ( Figures 5B  and 5D, green curve). Moreover, GST pull-down assays revealed only modest effects of R53S on binding to the eIF2b-NTT and eIF5-CTD ( Figure 5E, right). The CSP assay with 15 N-eIF1-R53S also demonstrates robust eIF5-CTD interaction with this mutant, as observed with WT 15 N-eIF1 ( Figure S7) (Reibarkh et al., 2008). Thus, R53S specifically abolishes eIF1 interaction with the eIF3c-NTD ( Figure 5B), which is not sufficient to impair accuracy of start site selection in vivo.
To demonstrate a role for eIF1-R53 in stabilizing the scanning PIC in vivo, we generated double mutants. Combining R53S and K56A in eIF1 did not alter the defect in eIF3c-NTD:eIF1 binding seen for R53S alone ( Figure 5B) and conferred only a moderate decrease in 40S:eIF1 binding affinity beyond the 11-fold reduction in K D induced by K56A alone (Figures 5B and 5D, blue and light green curves). Nevertheless, the R53S,K56A double mutant displayed a marked increase in UUG initiation that was not observed for single mutants ( Figure 6A, row 6). Since K56A has no effect on eIF1 binding to eIF2b-NTT and eIF5-CTD when (legend continued on next page) combined with four other eIF1 substitutions in a1 (M5 mutation) (Reibarkh et al., 2008), we conclude that the synthetic Suiphenotype of the R53S,K56A substitution ( Figure 6A) results from the combined loss of eIF1 interaction with eIF3c-NTD conferred by R53S and weakened 40S binding conferred by K56A ( Figure 5B). As shown in Figure 6B (panel 2, row 2 versus 4), the eIF1-R53S substitution also exacerbates the elevated UUG initiation caused by the eIF2b-S254Y variant (encoded by SUI3-2), previously attributed to increased GTP hydrolysis (Huang et al., 1997) and stabilizing the P IN conformation of Met-tRNA i at UUG codons (Martin-Marcos et al., 2014). Our findings imply that the defective stabilization of the closed/P IN conformation at UUG codons conferred by eIF2b-S254Y is normally mitigated by the eIF1/eIF3c-NTD interaction (disrupted by eIF1-R53S) to diminish acceptance of codon-anticodon mismatches in the P-site.
In conclusion, these results show that eIF1-R53 and -L96 are key eIF3c-NTD interaction sites in vivo. Within the scanning PIC, eIF3c-NTD appears to be the sole binding partner of eIF1-R53, whereas eIF1-L96 appears to engage both eIF3c-NTD and eIF5-CTD. Thus, multiple interactions between eIF3c, eIF5, and the ribosome collaborate in retaining eIF1 within the scanning PIC ( Figure 5A; also see Supplemental Results).
When bound to eIF3c-B 36-163 defective in eIF5-binding, eIF1 was unable to bind eIF5, and free eIF5 and the B 36-163 :eIF1 complex were found co-sedimenting at 3S ( Figure 7C Here, it should be noted that the eIF5-CTD:3c0 interaction precludes the 3c0:eIF1 interaction that otherwise competes with eIF1:40S association, and we propose that this stabilizes the scanning PIC. Based on these findings, we suggest approximate locations of the eIF5-CTD and eIF3c0 in the PIC ( Figure 7E) compatible with the proposed roles of these segments in regulating the transition from scanning to start codon recognition.

DISCUSSION
The results of NMR and complementary quantitative binding assays presented in this work revealed two distinct eIF1 complexes formed with overlapping eIF3c-NTD segments that appear to function at different stages of the initiation pathway. The C-terminal segment of the eIF3c-NTD (fragment D 59-163 ) (D) Schematic illustration of the proposed 4.6S trimeric complex. eIF3c-NTD is drawn as blue orange line representing unstructured segments, 3c0 (aa 1-58), and orange circles representing 3c1 (aa 59-87) and 3c2 (aa 105-159), as found in cryo-EM models in Figure 2 and redefined based on 15  , eIF5-CTD (dark green circle, this study) and 3c0 (aa 1-58) (orange line, this study) are superimposed onto the re-calculated cryo-EM structure, as shown in Figure 2B, right. See also Figure S8 and Table S1. contains the core eIF1-binding unit 3c1 (aa 59-87) and the adjacent globular domain 3c2 (aa 105-159), which bind to a limited surface on eIF1, including R53 and L96, in a manner compatible with eIF1 binding to the 40S subunit ( Figures 4A and 4C). We have assigned two densities projecting from the main body of eIF3 in the eIF1:eIF3:40S cryo-EM structure (Erzberger et al., 2014) as 3c1, which contacts eIF1, and 3c2 interacting with uS15 ( Figure 2). In contrast, eIF3c fragment C 36-87 , containing 3c1 and the C-terminal half of 3c0, interacts with a broader surface of eIF1 that includes R53 and L96 but additionally contains residues surrounding the two 40S binding sites at the C terminus of a1 and loop 1 (Figures 4C and 4B). Based on the Ssuphenotype of a mutation in Box6 (aa 51-60) within 3c0, we propose that interaction of eIF1 with 3c0 occludes the 40S-binding surface in eIF1 and thus facilitates eIF1 dissociation at the start codon-the event diminished at UUG codons by the Box6R Ssumutation. This destabilizing effect is likely to be opposed in the scanning PIC through eIF5-CTD binding to 3c0, which shifts eIF1 interaction from eIF3c-NTD elements 3c0/3c1 to 3c1/3c2 and thereby eliminates occlusion of the 40S binding surface on eIF1 by segment 3c0 ( Figure 7D). Dissolving the eIF5-CTD:3c0 interaction thus emerges as a key step in the transition from the open to closed conformation of the PIC, and we propose a plausible mechanism for this rearrangement below.
In agreement with our proposal that the 3c1/3c2 segments of eIF3c-NTD cooperate to anchor eIF1 on the scanning PIC, eIF1 substitution L96P, which perturbs the interface with 3c1, reduces eIF1 binding to the eIF3c-NTD. By also impairing eIF1 binding to the eIF5-CTD, L96P dramatically elevates UUG initiation in the manner expected for destabilization of the scanning complex (Martin-Marcos et al., 2013). eIF1 substitution R53S, which affects the neighboring surface in helix a1, dramatically reduces eIF3c-NTD binding but does not substantially impair the eIF1:eIF5-CTD interaction. Because R53S elevates UUG initiation only when combined with the a1 substitution K56A, which weakens eIF1:40S interaction, we conclude that a network of eIF1 interactions with the eIF3c-NTD, eIF5-CTD, and 40S subunit cooperate to anchor eIF1 to the scanning PIC and block initiation at non-AUG codons ( Figure 5A). Based on the cryo-EM model in Figure 2A, the role of 3c2 in anchoring eIF1 to the PIC appears to be indirect. Consistently, mutations altering Box12 Sui+ (aa 111-120) within 3c2 can elevate UUG initiation by either increasing or decreasing eIF1 retention in native PICs (Kará sková et al., 2012). This complexity may reflect dual role of 3c2 in promoting eIF1 binding to segment 3c1 and eIF5-CTD binding to 3c0 in the scanning PIC, while preventing the more stable eIF1 complex formed with 3c0/3c1 on AUG recognition. In addition, by directly contacting 40S protein uS15/S13, 3c2 is likely to stabilize eIF1 binding to the scanning PIC (Figure 2A Figures 8B and 8C). The 3c0 segment is now free to engage eIF1 and occlude its ribosome-binding surface, interfering with eIF1 re-association with the 40S subunit and thus allowing Met-tRNA i Met to remain stably anchored in the P IN state ( Figure 8D). These effects are expected to amplify the subtle distortion of eIF1 structure and perturbation of its 40S binding site that accompanies Met-tRNA i Met isomerization to the P IN state (Hussain et al., 2014). In this way, 3c0 ensures irreversible eIF1 release from the decoding center in response to AUG recognition and subsequent closure of the ribosome structure and formation of the 40S initiation complex.
It is noteworthy that human eIF1 also binds eIF3c-NTD (Fletcher et al., 1999) and eIF5-CTD (Luna et al., 2012). While the eIF3c-NTD segments corresponding to 3c0 are shorter in animals and plants, they contain an acidic element similar to Box6, lying next to the conserved core region 3c1 (Boxed in Figure S1B). Moreover, eIF3c-NTD in animals and plants is predicted to form an a-helical structure, as found in yeast 3c2 ( Figure 1C). Further work on the human and yeast systems is expected to reveal Eukarya-wide conservation of the MFC's role in promoting scanning and AUG selection through the coordinated interactions of the eIF3c-NTD with eIF1, eIF5 and potentially

EXPERIMENTAL PROCEDURES Protein Purification and Yeast Methods
Isotopically labeled or unlabeled proteins were expressed in E. coli transformants carrying appropriate plasmids (Table S1) and purified as described in Supplemental Information. Yeast Saccharomyces cerevisiae strains used in this study are constructed as described in Supplemental Information and listed in Table S2. Standard yeast molecular biology methods including growth and b-galactosidase assays were used throughout (Lee et al., 2007) (see Supplemental Experimental Procedures for details).
Biophysical Methods ITC, NMR spectroscopy, FA, and AUC are all performed as described in Supplemental Experimental Procedures. Detailed NMR data and structural statistics for eIF3c-B 36-163 and eIF1 are summarized in Tables S3 and S4, respectively. We re-ran integrative modeling prediction including the new information from the eIF3c-B NMR structure, with parameters and methods identical to those previously described (Erzberger et al., 2014).

ACCESSION NUMBERS
The accession numbers for the yeast eIF1 and eIF3c (aa. 36-163) data reported in this paper are PDB: 2rvh and 5H7U, respectively.     Left, Chemical shift perturbation (CSP), Δδ, was computed as described in Supplementary Materials and methods and presented for each assigned amino acid. "P"s indicate proline residues. Black boxes indicate the residues that were not assigned. Shaded regions indicate the residues whose signal in the 1 H- 15   Cartoon representation of one of the 500 solutions generated by our modeling runs. eIF3a is colored gold, eIF3c in orange, eIF1 in brown, rpS13/uS15 in cyan, rpS27/eS27 in blue and rpS1/eS1 in green. Interstrand crosslinks are shown in red. Additional structural elements present in the modeling have been omitted for clarity.         c For residues Asn24-Ile30, Leu39-Val69, Ile77-Phe108 of eIF1.

The effect of eIF1-K60E on eIF1 binding to its MFC partners
The K60E substitution strongly impairs 40S binding in vitro, thereby allowing mis-initiation from UUG codons in vivo (Suiphenotype) (3). Our GST pulldown assays indicated that K60E also disrupts eIF1 binding to eIF2β-NTT and the eIF5-CTD (Fig. 5E, col. 3); a defect in eIF5-CTD binding was confirmed by CSP assay with 15 N-eIF1-K60E ( Fig. S7). It is noteworthy that the K60E substitution essentially eliminates eIF1 binding to the 40S subunit (Figs. 5B and S6B) (3) as well as to the eIF2β-NTT and eIF5-CTD (Fig. 5B), and yet, confers a less dramatic increase in UUG initiation compared to L96P (Fig. 6A). One possibility is that eIF1-K60E's robust interaction with eIF3c-NTD ( Fig. 5B and E) is sufficient to prevent a more dramatic reduction in accuracy for this substitution in vivo. Another possibility in light of the proposed role for 3c0-Box6 Ssu+ in eIF1 release is that the inability of eIF1-K60 to bind eIF2β-NTT dampened inaccurate UUG initiation by stabilizing the open state, assuming that eIF2β-NTT contributes to eIF1 release by preventing eIF1 rebinding to the ribosome. These two possibilities must be distinguished by experiments in the future.