then separated on 10% polyacrylamide (8M urea) sequencing gel in 1xTBE buffer. b-galactosidase assays were conducted as described previously [13]. GST-pull-down experiments, preparation of whole-cell extracts, sucrose gradient separations and Western blot analysis of gradient fractions were essentially conducted as described in [39,40]. Supporting Information Figure S1 The 59 sequences of uORF1 contain at least three REI-promoting elements (RPEs), one of which operates in the a/ TIF32-NTD-dependent manner. (A) Schematic showing the solitary uORF1 GCN4-lacZ construct with the battery of deletions in the uORF1’s 59UTR defined below and used in panel B. (B) The YBS47 and YBS53 strains were introduced with the GCN4- lacZ deletion constructs described in panel A and analyzed as in Figure 2B. This panel is showing identical data to those presented in Figure 2B except that the obtained values were not expressed relative to the value obtained with the wt uORF1-GCN4-lacZ construct. (C) Quantitative RT-PCR of the selected GCN4-lacZ transcripts shows no significant changes in their stability. Primers matching the lacZ fusion gene were used for quantification. ADH1 was used as an internal normalization standard. Values obtained for individual mutant constructs in triplicates are expressed relative to the value obtained with the wt GCN4-lacZ construct p209. Error bars = SD. (EPS) Figure S2 The RPEs i., ii., and iv. of the 59 enhancer are critically required for up-regulation of GCN4 expression under starvation conditions. (A) Schematic showing the inducible uORF1 – uORF4 GCN4-lacZ construct with the selected substitutions and/or deletions in the color-coded RPEs that are used in panel B. (B) The YBS47 strain was introduced with the GCN4-lacZ deletion and/or substitution constructs described in panel A and analyzed as in Figure 1D. To induce the GCN4-lacZ expression, the transformants grown at the minimal media for 2 hrs after dilution were treated with 10 mM 3-AT for 6 hrs. wt#, construct 1111 (pM23); bg#, construct 4411 (pVM37). (EPS) Figure S3 The a/tif32-Box6 and Box17 mutations neither decrease the overall eIF3 affinity for 40S subunits in vivo nor reduce binding of the a/TIF32-NTD to the small ribosomal protein RPS0A in vitro. (A) Isogenic strains derived from YBS52 (GCN2 a/ tif32D) replacing the resident YCpTIF32-His-U plasmid by YCp- References 1. Jackson RJ, Hellen CUT, Pestova TV (2010) The mechanism of eukaryotic transla- tion initiation and principles of its regulation. Nat Rev Mol Cell Biol 11: 113–127. 2. Calvo SE, Pagliarini DJ, Mootha VK (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. PNAS 106: 7507–7512. 3. Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283–292. 4. Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS (2009) Genome- Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science 324: 218–223. 5. Ivanov IP, Loughran G, Atkins JF (2008) uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs. PNAS 105: 10079–10084. 6. Hood HM, Neafsey DE, Galagan J, Sachs MS (2009) Evolutionary Roles of Upstream Open Reading Frames in Mediating Gene Regulation in Fungi. Annual Review of Microbiology 63: 385–409. 7. Kozak M (2005) Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361: 13–37. 8. Hinnebusch AG (2005) Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol 59: 407–450. Translation Reinitiation Promoting Elements a/TIF32-His-screen, YCp-a/tif32-Box6-His or YCp-a/tif32- Box17-His, respectively, as described in Figure 5B were grown in YPD medium at 30uC to an OD600 of ,1–1.5 and cross-linked with 2% HCHO prior to harvesting. WCEs were sedimented through 7.5 to 30% sucrose gradients, collected fractions were pooled as indicated and subsequently subjected to Western analysis with antibodies against the denoted proteins. An aliquot of each WCE was analyzed in parallel (In, input). The amounts of each factor in the 43S fractions (boxed) obtained from three independent experiments were normalized for the RPS0A level and the ratios of the eIF/40S levels in the mutant to those in the WT were averaged. The means and standard errors are plotted in the histogram. (B) RPS0A fused to GST (lane 3) or GST alone (lane 2) were tested for binding to the 35S-labeled a/TIF32-NTD (amino acid residues 1–400) and its mutant derivatives in GST pull down assays. The GST proteins were visualized by Coomassie blue staining (top); radiolabeled proteins by autoradiography (bottom). Lane 1 contains 20% of the input amounts of corresponding in vitro translated proteins used in the individual binding reactions. (TIF) Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX) Text S1 (DOC) Yeast strains used in this study. Plasmids used in this study. Oligonucleotides used in this study. Supporting Results and Materials and Methods. Acknowledgments We are thankful to Thomas Dever and Monica Liu for critical reading of the manuscript, to Karel Mikul´ık for his excellent help with the RNA structure probing, to Jana Nejep´ınska´ for her help with qRT-PCR, to Olga Kry´dova´ for technical and administrative assistance, and to the members of the Vala´sˇek and Kra´sny´ laboratories for helpful suggestions. Author Contributions Conceived and designed the experiments: LSV VM JP. Performed the experiments: VM SG ID. Analyzed the data: LSV VM JP SG. Contributed reagents/materials/analysis tools: BS. Wrote the paper: LSV. Performed the computational analysis: JP. Performed most of the experiments: VM. 9. Kozak M (2001) Constraints on reinitiation of translation in mammals. Nucleic Acids Res 29: 5226–5232. 10. Szamecz B, Rutkai E, Cuchalova L, Munzarova V, Herrmannova A, et al. (2008) eIF3a cooperates with sequences 59 of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA. Genes Dev 22: 2414–2425. 11. Dever TE (2002) Gene-specific regulation by general translation factors. Cell 108: 545–556. 12. Vattem KM, Wek RC (2004) Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci U S A 101: 11269–11274. 13. Grant CM, Hinnebusch AG (1994) Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control. Mol Cell Biol 14: 606–618. 14. Grant CM, Miller PF, Hinnebusch AG (1995) Sequences 59 of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation. Nuc Acids Res 23: 3980–3988. 15. Vala´sˇek L, Mathew A, Shin BS, Nielsen KH, Szamecz B, et al. (2003) The Yeast eIF3 Subunits TIF32/a and NIP1/c and eIF5 Make Critical Connections with the 40S Ribosome in vivo. Genes Dev 17: 786–799. PLoS Genetics | www.plosgenetics.org 15 July 2011 | Volume 7 | Issue 7 | e1002137