SUPPLEMENTARY INFORMATION

Yeast strain constructions

Strain CHY01 was constructed by introducing HO URA3 plasmid YCp50-HO into strain H1895 for mating type switching. The resulting diploid was transformed with a sui1::hisG-URA3-hisG disruption cassette contained on pCFB01 to delete chromosomal SUI1. The diploid was than transformed with sc SUI1 LEU2 plasmid pCFB02. Sporulation and tetrad dissection were carried out to select a Ura+ Leu+ ascospore. Uracil auxotrophy was regained by growing the ascospore clone on 5-fluoro-orotic acid (5-FOA) plates, and the resulting strain was transformed with sc

SUI1 URA3 plasmid p1200. pCFB02 was eliminated by growing in YPD medium, to yield strain

CHY01. Strain JCY03 was constructed by crossing CHY01 with a Leu+ transformant of H2995 harboring YCplac111 and conducting sporulation and tetrad dissection to isolate Trp- Leu+ His-

Ura+ ascospores that are unable to grow on 5-FOA medium and are resistant to sulfometuron

(GCN2+). The strain was then plated on YPD to lose the LEU2 plasmid and thereby obtain strain

JCY03.

Plasmid constructions and site-directed mutagenesis.

Construction of plasmid pCFB01 containing the sui1::hisG-URA3-hisG disruption cassette was as follows. Sequences immediately 5’ of the SUI1 ORF were amplified by PCR using primers CHA28 (GCAGATCTGAATTCATTCTGGACATCCTG) and CHA29

(GGCGGTTTCGTCGGATCCTGTGTCGGCG), introducing EcoRI and BamHI restriction sites

(sequences underlined) at the ends of the fragment. Sequences immediately 3’ of the SUI1 ORF were amplified with primer CHA26 (CATTGTCAAGGATCCAGAAATGGGGG) and CHA27

(CAGATTCAAATCGGTCGACCCCATGATAATG), introducing BamHI and SalI restriction

1 sites at the ends. The PCR products were double-digested with the corresponding restriction

enzymes, vector pUC18 was double digested with EcoRI and SalI, and plasmid pNKY51 (Alani

et al. 1987) containing the hisG::URA3::hisG cassette was digested with BamHI (to liberate the

cassette on a BamHI fragment), and a ligation reaction was carried out with all four fragments to

produce pCFB01.

LEU2 plasmids p4389 and pCFB03 containing His-SUI1 were constructed as follows.

A PCR product was amplified from SUI1 plasmid p1200 using the “upstream” primer CHA22

(5’ CCGGGTCGGAAGCTTAAACCAGTGACAC) and the “downstream” primer CHA182 (5’

GATTTCAGATTCTCAATGGAGTGGTGGTGGTGGTGGTGCATACGATTTGCTTCAGCT

ATATTAATATATTCC), which contains a novel HindIII site present 5’ of the SUI1 ORF and

the complement of the coding sequences for the His6 tag immediately following the ATG . p1200 was also amplified with the upstream primer CHA183 (5’

GAAGCAAATCGTATGCACCACCACCACCACCACTCCATTGAGAATCTGAAATCATTT

GATCC) and downstream primer CHA27 (5’ CAG

ATTCAAATCGGTCGACCCCATGATAATG) to produce a fragment with the coding

sequences for the His6 tag and a novel SalI site downstream of the SUI1 ORF. The two PCR

products were joined by fusion PCR using primers CHA22 and CHA27 and the resulting

fragment was double-digested with HindIII and SalI and inserted between the corresponding

sites in hc LEU2 vector YEplac181 to make p4389, and into sc LEU2 vector YCplac111 to make

pCFB03.

Mutations were introduced into the His-SUI1 allele on p4389 or pCFB03 by PCR fusion

using the mutagenic primers listed in Table S2. For example, to construct pCFB101, plasmid

pCFB03 was amplified with primers CHA33 (GCTCACTCATTAGGCACCCCAGGC) and

2 CHA102 (Table S2) as well as with primers CHA101 (Table S2) and CHA34

(GTTGGGAAGGGCGATCGGTGCGG). The PCR products were joined by fusion PCR, double digested with HindIII and SalI and cloned into YCplac111, yielding pCFB101. The same strategy was employed to construct mutant plasmids pCFB129, pCFB130 and pCFB134. For mutant plasmids pJCB01 and pJCB03, the GeneTailorTM site-directed mutagenesis system

(Invitrogen) was employed with the corresponding primers listed in Table S2 and using pCFB03 as template. pJCB02 and pJCB04 were constructed by digesting pJCB01 and pJCB03 with

HindIII and XbaI and inserting the His-SUI1 fragments into YEplac181.

To construct plasmids for expressing recombinant His6-eIF1 from the T7 promoter, pCFB03, pCFB101 and pCFB129 were amplified with primer JCO-01

(CGCCGCCATATGCACCACCACCAC), to introduce a NdeI restriction site at the 5’ end of the ORF, and primer JCO-02 (CCCAAGCTTTTAAAACCCATGAAT), to introduce a HindIII restriction site at the 3’ end of the SUI1 ORF. The product was double digested with NdeI and

HindIII and cloned into the vector pT7-7 to obtain pJCB05, pJCB06 and pJCB07. pJCB08 was constructed in the same manner but primer JCO-02-2 (CCCAAGCTTTTAAAACCGATGAAT) was used instead of JCO-02.

Biochemical assays with yeast extracts

For measuring luminescence in WCEs of strains harboring pRaugFFuug, cells were lysed with

Passive Lysis Buffer (Promega) for 40-60 min and luminescence was measured with a BMG

FLUOstar OPTIMA 96 well plate reader. The instrument injects firefly luciferase buffer (15 mM Tris [pH 8.0], 25 mM glycylglycine, 4 mM EGTA, 2 mM ATP, 1 mM DTT, 15 mM

MgSO4, 0.1 mM CoA, 75 M luciferin, final pH adjusted to 8.0), measures luminescence, then

3 injects renilla luciferase buffer (0.22 M citric acid-sodium citrate buffer [pH 5.0], 1.1 M NaCl,

2.2 mM Na2EDTA, 0.44 mg/ml BSA, 1.3 mM NaN3, 1.43 M coelenterazine, final pH adjusted to 5.0), which quenches the firefly reaction and provides the substrate for the renilla reaction.

For analysis of polysome profiles and fractionation of native PICs from cross-linked cells,

WCEs were made from 300 ml of cells grown to an OD600 of 1.0 in SC medium. Cells were transferred to a 500 ml centrifuge tube containing 75 g of shaved ice and the tube was inverted five times. HCHO was added to 1% and the tube was inverted 10 times and left on ice for 1 h.

Glycine was added to 0.1 M and the cells were collected by centrifugation for 10 min at 7000 rpm in a Sorvall RC5B rotor. The pellet was resuspended in 7 ml of buffer B (20 mM Tris (pH

7.5), 50 mM KCl, 10 mM MgCl2) supplemented with EDTA-free protease inhibitor tablet

(Roche), 5 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride (PMSF). The cell suspension was transferred to a 15 ml conical tube and centrifugated for 5 min at 4200 rpm in a

Beckman J-6B centrifuge and the supernatant was decanted. One vol of cells was resuspended in

1.3 vol of buffer B and 1.3 vol of glass beads, and cells were lysed by vortexing eight times for

30 s with 30 s intervals on ice. The lysate was centrifugated for 5 min at 4200 rpm and the supernatant was transferred to an Eppendorf tube. The extract was cleared by two consecutive centrifugations at 13,000 rpm for 5 and 10 min in an Eppendorf 5415D centrifuge, collecting the supernatant while avoiding the lipid layer at the top and the pellet. The WCEs were separated by sedimentation through sucrose gradients as described previously (Asano et al. 2000). For analysis of 43S complexes, 700 μL fractions were collected, mixed with 6X loading dye [300 mM Tris-HCl pH 6.8, 12% (w/v) SDS, 0.6% (w/v) bromophenol blue, 60% (v/v) glycerol and

600 mM -mercaptoethanol] and boiled for 10 min to reverse the cross-links. 20 μL samples were separated on 4-20% Tris-Glycine polyacrylamide gels (Criterion, BioRad). After

4 electroblotting to nitrocellulose membranes, membranes were probed with polyclonal antibodies

against the appropriate initiation factors and 40S ribosomal proteins. Antibodies against eIF3b,

eIF5, eIF2 (Phan et al. 1998), eIF2 (Dever et al. 1992), eIF1A (Olsen et al. 2003) and eIF1

(Valasek et al. 2004) have been described. RPS2 antibody (generated in rabbit) was kindly

provided by Jon Warner. For resedimentation experiments, the conventional protocol was

followed except that twofold-greater A260 units of WCEs were resolved on the gradient and,

following the first sedimentation, the 40S fractions were pooled, diluted with 10 volumes of

buffer B supplemented with 1mM dithiothreitol, concentrated using an Amicon Ultra-4 centrifugal filter device (Millipore), and sedimented through a second sucrose gradient under the

same conditions used for the first gradient separation.

Biochemical assays in the reconstituted yeast system

Reagent preparation

Initiation factors eIF1, eIF1A, eIF5 and mutant variants of these proteins were purified using the

IMPACT system for purification of intein fusion proteins as described (Algire et al. 2002).

eIF1A and eIF1 were labeled using expressed ligation (Maag and Lorsch 2003). His-

tagged eIF2 was overexpressed in yeast and purified as described (Algire et al. 2002). 40S

subunits were purified as described previously (Algire et al. 2005). Model mRNAs of the

sequences 5-GGAA[UC]7UAUG[CU]10C-3 and 5-GGAA[UC]7UUUG[CU]10C-3 were

synthesized using T7 polymerase run-off transcription and purified by denaturing

polyacrylamide gel electrophoresis as described (Lorsch and Herschlag 1999). Yeast initiator

tRNA was synthesized from a hammerhead fusion template using T7 polymerase run-off

5 35 transcription, as described (Algire et al. 2005), and [ S]Met-tRNAi and stoichiometrically-

charged Met-tRNAi were prepared as described (Kapp and Lorsch 2004).

43S Complex Formation Assays

Gel shift assays were performed as described previously (Algire et al. 2002). Experiments

35 measuring the rate of TC binding were performed with limiting S-Met-tRNAi (0.5 nM) and

saturating GDPNP (1 mM), eIF2 (200 nM), eIF1 (1 M), eIF1A (1 M) and mRNA(AUG) (2

M) or mRNA(UUG) (50 M) and 10 nM 40S subunits. Reactions were stopped with a chase of

unlabeled ternary complex. This chase prevented additional labeled TC from forming 43S

complex, keeping the reaction from progressing. The chase was made at 6X concentration and

consisted of 6 mM GDPNP, 2.1 M eIF2, 900 nM unlabeled Met-tRNAi with buffer as described

above. Controls indicate that the chase is stable for many hours, and that labeled 43S complex is

stable for longer than one hour after chasing. A control in which the chase was added before the labeled TC demonstrated that the chase was effective.

FRET Experiments

FRET experiments were carried out as previously described (Maag et al. 2005) using a

SX.180MV-R stopped flow fluorometer from Applied Photophysics. One syringe contained mRNA (50 μM final) and an unlabeled eIF1 chase (3 μM final) in 1X reconstitution buffer. The other syringe contained 43S complex with final concentrations of 1mM GDPNP, 900 nM eIF2,

450 nM Met-tRNAi, 50 nM eIF1A-Fl, 60 nM eIF1-TAMRA and 150 nM 40S ribosomal subunits

in 1X reconstitution buffer. Buffer conditions were the same as those used in fluorescence

anisotropy experiments. The excitation wavelength used was 490 nm and a high-pass KV filter

6 with a cut-off of 515 nm was used to restrict the emission wavelengths. Donor fluorescence was

monitored as a function of time following rapid mixing. The data were fit to a double

exponential equation using non-linear regression. Fitting to a double exponential lowered the 2

value by more than three-fold and greatly reduced systematic errors. FRET experiments were

repeated 5-7 times.

References for Supplementary information

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7 Kawagishi-Kobayashi, M., J.B. Silverman, T.K. Ung, and T.E. Dever. 1997. Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2. Mol Cell Biol 17: 4146-4158. Lomakin, I.B., V.G. Kolupaeva, A. Marintchev, G. Wagner, and T.V. Pestova. 2003. Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing. Genes Dev 17: 2786-97. Lorsch, J.R. and D. Herschlag. 1999. Kinetic dissection of fundamental processes of initiation in vitro. EMBO J 18: 6705-6717. Maag, D., C.A. Fekete, Z. Gryczynski, and J.R. Lorsch. 2005. A Conformational Change in the Eukaryotic Translation Preinitiation Complex and Release of eIF1 Signal Recognition of the Start Codon. Mol Cell 17: 265-75. Maag, D. and J.R. Lorsch. 2003. Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit. J Mol Biol 330: 917-24. Olsen, D.S., S. E.M., A. Mathew, F. Zhang, T. Krishnamoorthy, L. Phan, and A.G. Hinnebusch. 2003. Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo. EMBO J 22: 193-204. Parent, S.A., C.M. Fenimore, and K.A. Bostian. 1985. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1: 83-138. Phan, L., X. Zhang, K. Asano, J. Anderson, H.P. Vornlocher, J.R. Greenberg, J. Qin, and A.G. Hinnebusch. 1998. Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Mol Cell Biol 18: 4935-4946. Russell, D. W., Jensen, R., Zoller, M. J., Burke, J.,Errede, B., Smith, M. and Herskowitz, I. 1986. Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region. Mol Cell Biol. 6: 4281-4294. Sikorski, R.S. and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27. Smith, D.B. and K.S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31-40. Tabor, S. and C.C. Richardson. 1987. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84: 4767-4771. Valasek, L., K.H. Nielsen, F. Zhang, C.A. Fekete, and A.G. Hinnebusch. 2004. Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection. Mol Cell Biol 24: 9437-55. Yoon, H.J. and T.F. Donahue. 1992. The sui1 suppressor locus in Saccharomyces cerevisiae Met encodes a translation factor that functions during tRNAi recognition of the start codon. Mol. Cell. Biol. 12: 248-260.

8 Table S1. Plasmids employed in this work.

Plasmid Description Source or reference YCplac111 sc LEU2 cloning vector (Gietz and Sugino 1988) YEplac181 hc LEU2 cloning vector (Gietz and Sugino 1988) YCp50-HO sc HO in YCp50 (Russell et al. 1986) YCp50 sc URA3 cloning vector p1200 sc URA3 SUI1 (3.3 kb EcoRV-HindIII fragment) (Yoon and Donahue in YCp50 1992) pNKY51 hisG-URA3-hisG disruption cassette in YEp24 (Alani et al. 1987) pCFB01 sui1::hisG-URA3-hisG disruption cassette in this study pUC18 pCFB02 sc LEU2 SUI1 in YCplac111 this study pCFB03 sc LEU2 His-SUI1 in YCplac111 this study pCFB04 hc LEU2 SUI1 YEplac181 this study p4389 hc LEU2 His-SUI1 in YEplac181 this study pCFB101 sc LEU2 His-SUI1-FDPF9,12ADPA in this study YCplac111 pCFB129 sc LEU2 His-SUI1-ISQLG93-97ASQAA in this study YCplac111 pCBF130 hc LEU2 His-SUI1-ISQLG93-97ASQAA in this study YEplac181 pCBF134 hc LEU2 His-SUI1-G107R in YEplac181 this study pJCB01 sc LEU2 His-SUI1-D83G in YCplac111 this study pJCB02 hc LEU2 His-SUI1-D83G in YEplac181 this study pJCB03 sc LEU2 His-SUI1-Q84P in YCplac111 this study pJCB04 hc LEU2 His-SUI1-Q84P in YEplac181 this study p1780-IMT hc URA3 SUI2, SUI3, GCD11, IMT4 in YEp24 (Asano et al. 1999) YEp24 hc URA3 cloning vector (Parent et al. 1985) p180 sc URA3 GCN4-lacZ with WT leader in YCp50 (Hinnebusch 1985) p367 lc URA3 HIS4(AUG)-lacZ (Donahue and Cigan 1988) p391 lc URA3 HIS4(UUG)-lacZ (Donahue and Cigan 1988) YCpSUI5- sc URA3 SUI5-G31R in YCplac33 (Valasek et al. 2004) G31R-U YCplac33 sc URA3 cloning vector (Gietz and Sugino 1988) pRS316(2.8kb) sc URA3 TIF5 in pRS316 (Chakrabarti, A and Maitra, U 1991) pRS316 sc URA3 cloning vector (Sikorski and Hieter 1989) pGEX-NIP1-N GST-NIP1-N fusion plasmid in pGEX-4T-1 (Asano et al. 2000)

9 Plasmid Description Source or reference pGEX-4T-1 GST expression vector (Smith and Johnson 1988) pT7-7 Cloning vector with T7 promoter (Tabor and Richardson 1987) pJCB05 His-SUI1 in pT7-7 this study pJCB06 His-SUI1-FDPF9,12ADPA in pT7-7 this study pJCB07 His-SUI1-ISQLG93-97ASQAA in pT7-7 this study pJCB08 His-SUI1-G107R in pT7-7 this study pRaugFFuug Dual luciferase reporter LUCren(aug)-LUCfirefly this study (uug) in URA3 vector

10 Table S2. Primers used for SUI1 mutagenesis (with mutated positions underlined)

Name Sequence Mutation CHA101 CCATTGAGAATCTGAAATCAGCAGATCCTGCCGC F9A, F12A CGACACAGGAGACGACG

CHA102 CGTCGTCTCCTGTGTCGGCGGCAGGATCTGCTGAT F9A, F12A TTCAGATTCTCAATGG

CHA129 GAGCTAAGGTTTGCGAATTTATGGCTTCCCAAGCT ISQLG93- GCATTACAAAAGAAGAACATTAAGATTCATGGG 97ASQAA

CHA130 CCCATGAATCTTAATGTTCTTCTTTTGTAATGCAG ISQLG93- CTTGGGAAGCCATAAATTCGCAAACCTTAGCTC 97ASQAA

CHA133 GGGATTGCAAAAGAAGAACATCAAGATTCATAGA G107R TTTTAAGTTCAAGGCTTACG

CHA134 CGTAAGCCTTGAACTTAAAATCTATGAATCTTGAT G107R GTTCTTCTTTTGCAATCCC

JCO-69 AGATTATTCAGTTGCAGGGTGGCCAAAGAGCAA D83G

JCO-70 ACCCTGCAACTGAATAATCTCCCCCATTTC D83G

JCO-71 TTATTCAGTTGCAGGGTGACCCAAGAGCAAAGG Q84P

JCO-72 TTATTCAGTTGCAGGGTGACCCAAGAGCAAAGG Q84P

11 Table S3. Yeast strains employed in this work.

Strain Genotypes Source H1895 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 (TRP1 GCN4-lacZ) (Kawagishi- Kobayashi et al. 1997) H1642 MATa ura3-52 leu2-3 leu2-112 trp163 (TRP1 GCN4-lacZ) (Dever et al. 1992) CHY01 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG ( TRP1 this study GCN4-lacZ ) p1200 (sc URA3 SUI1) H2995 MAT ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) this study JCY03 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study p1200 (sc URA3 SUI1) JCY103 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 GCN4-lacZ) pCFB03 (sc LEU2 His-SUI1) JCY105 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) p1200 (sc URA3 SUI1) p4389 (hc LEU2 His-SUI1) JCY107 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) p4389 (hc LEU2 His-SUI1) JCY115 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pCFB101 (sc LEU2 His-SUI1-FDPF9,12ADPA) JCY137 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pCFB129 (sc LEU2 His-SUI1-ISQLG93-97ASQAA) JCY141 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pCFB130 (hc LEU2 His-SUI1-ISQLG93-97ASQAA) JCY211 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) p1200 (sc URA3 SUI1) pCFB134 (hc LEU2 His-SUI1- G107R) JCY217 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pJCB01 (sc LEU2 His-SUI1-D83G) JCY221 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pJCB02 (hc LEU2 His-SUI1-D83G) JCY225 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pJCB03 (sc LEU2 His-SUI1-Q84P) JCY229 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pJCB04 (hc LUE2 His-SUI1-Q84P) JCY253 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) pCFB02 (sc LEU2 SUI1) JCY255 MATa ura3-52 leu2-3 leu2-112 trp163 gcn2 sui1::hisG (TRP1 this study GCN4-lacZ) p1200 (sc URA3 SUI1) pCFB04 (hc LEU2 SUI1) JCY145 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pCFB03 (sc LEU2 His-SUI1) JCY149 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study p4389 (hc LEU2 His-SUI1)

12 Strain Genotypes Source JCY189 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pCFB129 (sc LEU2 His-SUI1-ISQLG93-97ASQAA) JCY193 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pCFB130 (hc LEU2 His-SUI1-ISQLG93-97ASQAA) JCY197 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pCFB134 (hc LEU2 His-SUI1-G107R) JCY237 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pJCB01 (sc LEU2 His-SUI1-D83G) JCY241 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pJCB02 (hc LEU2 His-SUI1-D83G) JCY245 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pJCB03 (sc LEU2 His-SUI1-Q84P) JCY249 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pJCB04 (hc LEU2 His-SUI1-Q84P) JCY261 MATa ura3-52 leu2-3 leu2-112 trp163 his4-303(AUU) sui1::hisG this study pCFB02 (sc LEU2 SUI1)

13 Supplementary Figure Legends

Figure S1 (A) Immunoblot analysis of eIF1 proteins in WCEs. (i) The gcn2 strains described in Fig. 1 containing the indicated His-SUI1 alleles on sc or hc plasmids were grown in

SC-L medium and WCEs were subjected to Western analysis with antibodies against eIF1 or

GCD6. Two different amounts of each extract (1X and 2X) differing by a factor of 2 were loaded in successive lanes. (ii) same as above except using gcn2 strains containing untagged SUI1+ on

a sc plasmid in addition to the WT or G107R alleles of His-SUI1 on a hc plasmid, and antibodies

against the His6 epitope were employed to detect eIF1-His6 proteins. (iii) same as (i) except that

GCN2 his4-303 strains described in Fig. 3, with WT or 93-97 alleles of His-SUI1 on sc or hc

plasmids, were grown in SC-L medium at 37°C. (iv) same as (i) except using GCN2 his4-303

strains described in Fig. 3 with the indicated His-SUI1 alleles and WCEs obtained by the TCA

extraction method. (B-C) The 9,12 mutation in His-SUI1 does not detectably reduce eIF

association with native PICs. (B) The gcn2 strains described in Fig. 1 with sc WT or sc 9,12

His-SUI1 alleles were grown in SC-L medium and native PICs extracted from cross-linked cells

were examined as in Fig. 2(A-B). (C) Levels of initiation factors in the 40S fractions were

quantified as in Fig. 2C. Results plotted are means ± SEs (n=3). (D-E) The G107R mutation in

His-SUI1 reduces eIF5 association with native PICs. (D) The GCN2 his4-303 strains

described in Fig. 3 with hc WT or hc G107R His-SUI1 alleles were grown in SC-L medium and

native PICs extracted from cross-linked cells were examined as described in Fig. 2(A-B). (E)

Levels of initiation factors in the 40S fractions were quantified as in Fig. 2C. Results plotted are

means ± SEs (n=3). (F) Amino acid starvation of wild-type cells does not evoke a strong

decrease in 40S bound eIF2. The GCN2 his4-303 strain with WT His-SUI1 was grown in SC-

LIV for 2h and 0.5 μg/ml sulfometuron (SM) was added to one-half of the culture to induce

14 translation of GCN4, as in Fig. 1D, and native PICs extracted from cross-linked cells were

examined as described in Fig. 2(A-B).

Figure S2 Overexpression diminishes the elevated initiation at UUG start codons in vivo

conferred by SUI1 mutations D83G, Q84P and 93-97. GCN2 his4-303 strains with the

indicated His-SUI1 alleles harboring the dual luciferase reporters LUCRenilla (AUG) and LUCfirefly

(UUG) on sc URA3 plasmid pRaugFFuug were grown in SC-UL medium for ~2 doubling times

to an OD600 of ~0.8 and the luminescence was measured in WCEs. The ratio of luminescence, in

relative light units, for the UUG to AUG reporter was calculated and plotted. Two independent

transformants (-a and –b) were analyzed for the sc and hc Q84P strains.

Figure S3 (A) The 93-97 mutation impairs eIF1 binding to the N-terminal domain of yeast eIF3c/NIP1 in vitro. A fusion of GST to residues 1-156 of eIF3c/NIP1 (NIP1-N) or GST alone were expressed in E. coli, immobilized on glutathione-Sepharose beads and incubated with [35S]-

methionine labeled WT or mutant (9,12; 93-97 or G107R) synthesized in rabbit reticulocyte lysates. The beads were washed and the bound proteins eluted, separated by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. “In” shows 20% of the input amounts of in vitro translated proteins added to each reaction. (B) The 93-97 mutation reduces binding of

MFC components to native PICs. The GCN2 his4-303 strains described in Fig. 3 with sc WT or sc 93-97 His-SUI1 alleles were grown in SC-L medium at 37°C and native PICs extracted from cross-linked cells were examined as described in Fig. 2(A-B). (C) Overexpression of the 93-97

His-SUI1 allele rescues association of MFC components with polysomes. GCN2 his4-303 strains with sc WT, sc 93-97 or hc 93-97 alleles of His-SUI1 were grown in SC-L medium at

15 37°C, and association of eIFs with polysomes was examined in extracts of cross-linked cells

resolved as in Fig. 2A. (D) Overexpression of the Q84P His-SUI1 allele restores association

of eIF1 with native PICs. GCN2 his4-303 strains with sc WT, sc Q84P or hc Q84P were grown

in SC-L medium and eIF1 association with native PICs extracted from cross-linked cells was

examined as described in Fig. 2(A-B).

Figure S4 Predicted positions of residues altered by Sui- mutations in eIF1 in a structural

model of eIF1 bound to the small ribosomal subunit. The locations of residues in human eIF1

homologous to those altered by the SUI1 mutations described here are colored in the space-

filling model of eIF1 bound to a prokaryotic 30S/[P-site tRNA]/mRNA complex constructed

previously (Lomakin et al. 2003). 93-97: magenta; G107R: orange; D83G: turquoise and Q84P:

navy. Mutant 9,12 in the unstructured N-terminal tail is not shown. tRNA: light brown ribbon; mRNA in A- and P-sites: red; 16S rRNA: gray ribbon; ribosomal proteins: light blue ribbons.

16 His-SUI1 alleles A (i) (ii) His-SUI1 alleles sc hc hc WT hc G107R WT 9,12 93-97 WT 93-97 /WT /WT 1X 2X 1X 2X 1X 2X 1X 2X 1X 2X 1X 2X 1X 2X eIF1 His GCD6 GCD6

(iii) (iv) His-SUI1 alleles His-SUI1 alleles o o 30 C 37 C sc hc WT 93-97 WT 93-97 WT D83G Q84P WT D83G Q84P 1X 2X 1X 2X 1X 2X 1X 2X 1X 2X 1X 2X 3X 1X 2X 3X 1X 2X 1X 2X 3X 1X 2X 3X eIF1 eIF1 GCD6 GCD6

B C Top sc WT Top sc 9,12 In In eIF3b

T ) 1.0 0.8 eIF2 0.9 γ 0.9 eIF2α 1

eIF5 2 : s c W eIF1A ( s c 9 , 1

RPS2 A b u n d a c e i 4 0 S 0 eIF1 eIF3b eIF2γ eIF5 eIF1A 40S 40S

D E Top hc WT Top hc G107R 4.9 In In 1.3 T ) 5 eIF3b 0.8 eIF2γ 1 eIF5 0.6 RPS2 eIF1 A b u n d a c e i 4 0 S 40S ( h c G 1 0 7 R : W 0 40S eIF3b eIF2γ eIF5 RPS2

F Top Top Induced WT In Uninduced WT In eIF3c

eIF2γ eIF2α eIF5

RPS2 eIF1 40S 40S

GENESDEV/2007/075226 Cheung_FigS1 0.60 0.6 0.57 0.53 0.52

0.43

0.36 0.36 0.31 0.3

0.22

o i t a r G U A / G U U n o i s s e r p x e C U L 0.04

0

SC WT sc D83Ghc D83Gsc 93-97hc 93-97hc G107sRc Q84Ps-ac Q84Ph-bc Q84Ph-ac Q84P-b

GENESDEV/2007/075226 Cheung_FigS2 A

-NIP1-N -NIP1-N -NIP1-N -NIP1-N

In GST GST In GST GST In GST GST In GST GST [35S]-eIF1

WT 9,12 93-97 G107R

B sc WT sc 93-97 In Top In Top

eIF3b eIF2γ eIF2α eIF5 RPS2 eIF1

40S 40S

C Top sc WT Top sc 93-97 Top hc 93-97

eIF3b eIF2γ eIF5 RPS2 eIF1

40S 80S Polysomes 40S 80S Polysomes 40S 80S Polysomes

D sc WT sc Q84P hc Q84P In Top In Top In Top eIF1 RPS2 40S 40S 40S

GENESDEV/2007/075226 Cheung_FigS3 G107R D83G Q84P 93-97

GENESDEV/2007/075226 Cheung_FigS4