, For protein purification, the genes encoding various yeast S. cerevisiae eIF proteins (wild-type or mutant) or their segments were amplified by PCR using budding yeast genomic DNA. The PCR products were digested with BamHI and NotI and then ligated into suitably cut, modified pET28b vector, Plasmid construction -Plasmids used in this study are listed in Table S1 and new plasmids were constructed as follows
, ATG, hexa-histidine tag or GST tag with a tobacco etch virus (TEV) protease cleavage site had been cloned between XbaI and BamHI restriction sites immediately upstream of the protein coding region (pET28-His or pET28-GST derivatives). pET28b derivatives expressing untagged eIF1 or its mutants were also constructed. For yeast genetics, YCpL-SUI1 (eIF1) derivatives carrying eIF1 R53 and K56 mutations (used in Fig. 6) were constructed by subcloning the 0.2-kb NdeI-BamHI fragment containing the N-terminal half of eIF1-coding region of the corresponding derivatives of pET28
, and YDpW-SUI3-2 were constructed by transferring 1.9-kb NotI-SalI fragment of YDpU-SUI3 and YDpU-SUI3
, Yeast strains and methods -Yeast strains used in this study are listed in Table S2. Derivatives of strain KAY1057
, Sui -mutations allow the his4-301 allele to express using its third codon, UUG, as the start codon. Thus, yeast his4-301 strains carrying eIF1 mutant alleles (used in Fig. 6A and listed in Table S2) were assayed for histidine auxotrophy. 5 µl of 1.5 A 600 units of these strains and their 10-fold serial dilutions are spotted onto synthetic complete (SC) medium lacking leucine (panel 1, +His) or the same medium except with trace histidine (3 µM; panel 2, -His) and incubated for 2 and 10 days, respectively. The growth in the -His plate depends on UUG-initiated translation of a histidine enzyme, and hence a phenotypic measurement of UUG initiation, To quantify UUG initiation frequency compared to AUG initiation, we transformed the eIF1 mutant strains with URA3 HIS4 AUG and his4 UUG -lacZ fusion plasmids and assayed for ?-galactosidase, as described
, Likewise, to evaluate the combined effect of eIF1-R53S and eIF2?-S264Y (in Fig. 6B), overnight cultures of transformants of KAY1057 (WT eIF1) or KAY1071 (eIF1-R53S) carrying YDpSUI3 (WT eIF2?) or
, YDpSUI3-2 (eIF2?-S264Y) were spotted similarly onto SC medium lacking uracil (panel 1, +His) or the same medium except with trace histidine (1 µM; panel 2, -His) and incubated for 2 and 9 days, respectively. For lacZ reporter assays, we used the KAY1057 and KAY1071 transformants carrying YDpW-SUI3-2 (SUI3-2 TRP1) and URA3 HIS4 AUG or his4 UUG -lacZ fusion plasmids and assayed
, GST-eIF2?-NTT 1-140 , and GST-eIF5-CTD 241-405 were allowed to bind 35 S-labeled eIF1 or its mutant species. The percentage of 35 S-eIF1 species pulled down with GST fusion proteins was quantified using a phosphoriager. Alternatively, we used WT and R53S versions of recombinant eIF1 expressed in bacteria for the pulldown assay. In this figure, the values for the binding of each eIF1 mutant are presented relative to those obtained with WT eIF1. Other experiments shown in Fig. 1B, 7A, S1B-C and S8 were done with GST-eIF3c-NTD derivatives and eIF1 and eIF5 that were expressed and purified in E. coli. In the case of Fig. 7A, we used an untagged, GST-pulldown assays -GST-pulldown assays were performed as described previously (12). In Fig. 3E, GST-eIF3c-N 1-156
, The proteins were expressed in LB medium overnight at 15°C after induction with 0.5 mM IPTG. For His-tagged proteins, harvested cells were re-suspended in Ni-NTA binding buffer (20 mM Tris pH 8.0, 500 mM NaCl, 100 mM urea, 25 mM imidazole and 10 mM ?-mercaptoethanol) and lysed using EmulsiFlex homogenizer (Avestin), Expression and purification of proteins -The pET28-His(TEV) plasmids encoding the desired proteins were employed for transformation of BL21(DE3)RIPL CodonPlus strain (Stratagene)
, Proteins were eluted by a 0-10 mM linear gradient of reduced glutathione. Peak fractions were incubated overnight with His-tagged TEV protease at room temperature while dialyzing against Ni-NTA low salt buffer (20 mM Tris pH 8.0, 100 mM NaCl, 25 mM imidazole and 10 mM ?-mercaptoethanol)
, 28 subsequent 10 µl injections every 210 seconds. For iTC200, each titration typically consisted of a preliminary 0.4 µl injection followed by 19 subsequent 2 µl injections every 150 seconds. All of the experiments were performed at 20°C. Data for the preliminary injection, which are affected by diffusion of the solution from and into the injection syringe during the initial equilibration period
, Spectral analysis was performed with KUJIRA 0.984 (14), a program suite for interactive NMR analysis working with NMRView (15), according to the methods described previously (16). The backbone and side chain 1 H, 15 N and 13 C resonances of the proteins were assigned by standard double-and triple-resonance NMR experiments (17, 18), and were deposited in the BioMagResDB (BMRB accession numbers 11599 for eIF1 and 11600 for eIF3c-B), eIF3c-B 36-163 were each concentrated to 0.4 mM in 20 mM sodium phosphate buffer, vol.80
, The 20 conformers that were most consistent with the experimental restraints were then used for further analyses. The final structures were validated and visualized by using the PROCHECK-NMR (23) and CHIMERA (24, 25). Detailed experimental data and structural statistics are summarized in Table S3 and S4. The final ensembles of 20 conformers were deposited in the Protein Data Bank, Structure calculations -Structure calculations of eIF1 and eIF3c-B 36-163 were performed using CYANA, pp.19-21
, containing 150 mM NaCl and 1 mM DTT. A series of 2D 1 H-15 N HSQC spectra were recorded for the samples containing 70 µM, Chemical shift perturbation experiments -All the proteins were dissolved in 20 mM sodium phosphate buffer
, Chemical shift perturbation (CSP), ??, was defined as ?? = [(?? H ) 2 +(?? N /6.5) 2 ] 1/2 , where ?? H and ?? N are the chemical shift differences for H N and 15 N, respectively, vol.27
, This helped us verify the perturbation of ~ 10 overlapping chemical shifts. In the 15 N-eIF1 CSP experiments with eIF5-CTD, we also used eIF5 242-396 lacking the C-terminal tail. The CSP observed with this segment was indistinguishable with that observed with eIF5 242-405 (Fig. 1E, left)
, Fluorescence Anisotropy Experiments -Initiation factors eIF1A and eIF1 WT and mutant variants of this protein were purified using the IMPACT system (New England Biolabs) as described before (28) using the appropriate pTYB2-derived constructs. eIF1 WT and mutant proteins were labeled at their C termini with cysteine-lysine-fluorescein dipeptide, using the expressed protein ligation system as described previously (29)
, Fluorescence anisotropy measurements of equilibrium binding constants (Kd) were performed using a T-format
, The excitation and emission wavelengths were 497 and 520 nm, respectively. The data were fit with a hyperbolic binding equation describing the binding of fluorescently labeled eIF1 mutants to 40S subunits to give Kd values (29)
, using an Optima XL-I analytical ultracentrifuge equipped with two optical systems, the Rayleigh interference and absorbance systems (Beckman Coulter). For centrifuge, we used an An-50 Ti rotor featuring cells with a standard 12-mm charcoal-epon double sector centerpiece and sapphire windows. Proteins were diluted with the AUC buffer to a final concentration of 20-25 uM each, Analytical ultracentrifugation (AUC) -AUC sedimentation velocity experiments were carried out in the AUC buffer (20 mM sodium phosphate, pH 7.0, 150 mM NaCl)
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