SUPPLEMENTAL DATA RESULTS

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1 SUPPLEMENTAL DATA RESULTS Ded1-mediated ribosomal scanning is less leaky than scanning promoted by eifs 4A/4B/4F. The efficiency of leaky scanning in the presence of Ded1 or of eifs 4A/4B/4F was investigated on mrnas containing two AUG triplets. The 5 -UTRs of these mrnas consisted of CAA repeats and therefore lacked potential near-cognate initiation codons, the upstream AUG triplets were in different nucleotide contexts, and the second AUG triplet was followed by a GUS reporter gene (Pestova and Kolupaeva, 2002; Pisarev et al., 2006; Supplemental Fig. 1A). The AUGgood/good triplet was flanked by purines at [-3] and [+4] positions, AUGgood/bad had a [- 3] purine and a [+4] pyrimidine, AUGbad/good had a [-3] pyrimidine and a [+4] purine, and the AUGbad/bad triplet was flanked by [-3] and [+4] pyrimidines. 48S complex formation was assayed in the absence and in the presence of eif1, which enables 43S complexes to discriminate against AUG codons with a nucleotide context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at -3 and +4 positions (bold) are most important (Pestova and Kolupaeva, 2002; Pisarev et al., 2006). In the absence of eif1, 48S complexes formed exclusively on the first AUG triplets irrespective of their nucleotide context in the presence of either Ded1 or eifs 4A/4B/4F (Figs. 3B-E, lanes 2 and lanes 4), but the efficiency of 48S complex formation in the presence of Ded1 (lanes 4) was much lower than in the presence of eifs 4A/4B/4F (lanes 2). In the presence of eif1, the overall efficiencies of 48S complex formation mediated by Ded1 and by eifs 4A/4B/4F were very similar (Supplemental Figs. 1B-E, compare lanes 3 with lanes 5), which is consistent with the dependence of Ded-1-mediated 48S complex formation on the simultaneous presence of eif1 and eif1a that was observed on Stem-1 and Stem-2 mrnas (Figs. 2A, 2C and 3C of the main text). However, although the general nucleotide context rule was maintained in the presence of Ded1, and the relative efficiency of initiation on the AUGgood/good codon compared to initiation on the AUG codon of the GUS reporter gene (Supplemental Fig. 1B, lane 5) was higher than in the case of the AUGbad/bad codon (Supplemental Fig. 1E, lane 5), scanning in the presence of Ded1 was considerably less leaky on all four mrnas with relatively fewer complexes formed on the AUG codon of the GUS reporter gene compared to the upstream AUGs (Supplemental Figs. 1B-E, lanes 3 and 5).

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3 FIGURE LEGENDS Supplemental Figure 1. Ribosomal scanning in the presence of Ded1 is less leaky than in the presence of eifs 4A/4B/4F. (A) Sequences of the 5 UTR of (CAA)n-AUGgood/good-GUS, (CAA)n-AUGgood/bad-GUS, (CAA)n-AUGbad/good-GUS and (CAA)n-AUGbad/bad-GUS mrnas (Pestova and Kolupaeva, 2002; Pisarev et al., 2006) with initiation codons in bold. Context nucleotides from 3 to +4 positions relative to the AUG codon are underlined. (B-E) Toe-printing analysis of 48S complex formation on mrnas (shown in A) in the presence of Ded1 and initiation factors as indicated. The positions of initiation codons, of the full-length cdnas and of assembled 48S complexes are shown on the sides of each panel. Lanes C/T/A/G depict corresponding DNA sequences. MATERIALS AND METHODS Purification of initiation factors, ribosomal subunits and aminoacylation of initiator trna Mammalian 40S and 60S ribosomal subunits, eif2, eif3, eif4f and eif5b were purified from rabbit reticulocyte lysate (RRL) (Pisarev et al., 2007). Yeast 40S ribosomal subunits were purified as described (Galkin et al., 2007). Recombinant eif1, eif1a, eif4a, eif4b, ΔeIF4G , eif5 and E. coli methionyl trna synthetase were expressed in E. coli BL21 (DE3) and purified as described (Pisarev et al., 2007). In vitro transcribed trna Met i was aminoacylated using recombinant E. coli methionyl trna synthetase (Lomakin et al., 2006). Purification of Ded1, DDX3 and DHX29 Recombinant N-terminally His-tagged Ded1 and DDX3 were expressed in E. coli BL21 (DE3) (Franca et al., 2007; Iost et al., 1999). Ded1 and DDX3 were purified by affinity chromatography on Ni-NTA-agarose followed by FPLC on a MonoQ HR5/5 column, from which they eluted at ~280 and ~260 mm KCl, respectively. Native DHX29 was purified from RRL (Pisareva et al., 2008). Analysis of ribosomal binding of Ded1 To investigate ribosomal association of Ded1, 90 pmol Ded1 were incubated with 30 pmol yeast 40S subunits, or a combination of 30 pmol 40S subunits from RRL, 60 pmol eif2, 60 pmol eif3, 120 pmol eif1, 120 pmol eif1a and 60 pmol Met-tRNA Met i in 400 µl reaction mixtures

4 containing buffer A + 1 mm ATP and 0.4 mm GTP at 37 o C for 10 minutes. To investigate the potential influence of Ded1 on ribosomal binding of DHX29, 40 pmol DHX29 were added to preincubated reaction mixtures containing Ded1, 40S subunits from RRL, eifs 2/3/1/1A and MettRNA Met i and incubated for another 10 minutes. All reaction mixtures were subjected to centrifugation through 10%-30% SDGs prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 1h 15min. Fractions that corresponded to ribosomal complexes were analyzed by SDS- PAGE with subsequent fluorescent SYPRO (Molecular Probes) staining or western blotting using Ded1 antibodies (Liu et al., 2002). REFERENCES Franca R, Belfiore A, Spadari S, Maga G (2007) Human DEAD-box ATPase DDX3 shows a relaxed nucleoside substrate specificity. Proteins 67: Galkin O, Bentley AA, Gupta S, Compton BA, Mazumder B, Kinzy TG, Merrick WC, Hatzoglou M, Pestova TV, Hellen CU, Komar AA (2007) Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5. RNA 13: Iost I, Dreyfus M, Linder P (1999) Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase. J Biol Chem 274: Liu HY, Nefsky BS, Walworth NC (2002) The Ded1 DEAD box helicase interacts with Chk1 and Cdc2. J Biol Chem 277: Lomakin IB, Shirokikh NE, Yusupov MM, Hellen CU, Pestova TV (2006) The fidelity of translation initiation: reciprocal activities of eif1, IF3 and YciH. EMBO J. 25: Pestova TV, Kolupaeva VG (2002) The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection. Genes Dev 16:

5 Pisarev AV, Kolupaeva VG, Pisareva VP, Merrick WC, Hellen CU, Pestova TV (2006) Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex. Genes Dev 20: Pisarev AV, Unbehaun A, Hellen CU, Pestova TV (2007) Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol. 430: Pisareva VP, Pisarev AV, Komar AA, Hellen CU, Pestova TV (2008) Translation initiation on mammalian mrnas with structured 5'UTRs requires DExH-box protein DHX29. Cell 135: