Immunoprecipitation experiments Cells were grown in 50 ml of YPD to an OD600 of 0.5, collected and washed with phosphate-buffered saline. Extracts were prepared in 500 ml of Buffer A (50 mM Tris pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 0.1% NP-40, 5 mM b-mercaptoethanol and protease inhibitors (Roche)) using Zirconia beads as described earlier (Granneman et al, 2009). RNA extractions and northern blot analyses were performed as described earlier (Tollervey, 1987) using oligonucleo- tides listed in Supplementary Table 2. Supplementary Figure 1 shows the regions to which the oligonucleotides hybridize. In vitro chemical modiﬁcation of puriﬁed pre-40S complexes One litre of BY4741 cells containing the pADH-HTP-Nob1 plasmid (a generous gift from Brigitte Pertschy) was grown to an OD600 of 0.5. Extracts were prepared in 4 ml of Buffer B (50 mM Hepes–KOH pH 7.5, 100 mM NaAc, 5 mM MgCl2, 5 mM b-mercaptoethanol, 0.1% NP-40 and protease inhibitors (Roche)). For each puriﬁcation, 1 ml of extract was incubated with 50 ml of IgG beads for 1 h at 41C. IgG beads were washed three times with Buffer B and resuspended in 50 ml of Buffer B for chemical modiﬁcation reactions. DMS modiﬁcation was performed in Buffer B for 10 min at 301C using 10 ml of 20% DMS (diluted into ethanol). The reaction was quenched by addition of 40 ml of mix 0.5 M b-mercaptoethanol and 0.5 M sodium acetate pH 5.5. One-methyl-7-nitroisatoic anhydride (1M7; Fisher Scientiﬁc) modiﬁcation reactions were performed in Buffer A at 301C for 1 min in the presence of 8 mM 1M7. CMCT modiﬁcation reactions were performed in Buffer C (50 sodium borate pH 7.5, 100 KCl, 5 mM MgCl2), for 10 min at 301C using 30 ml of 80 mg/ml CMCT, freshly prepared in water. To map the modiﬁed nucleotides, primer extension reactions were per- formed using oligonucleotide ITS1 RT (Supplementary Table 2) as described earlier (Granneman et al, 2009) using B100 ng of puriﬁed RNA or 1 mg of total RNA. 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Modelling of RNA-binding sites To visualize the RNA-binding sites in the S. cerevisiae 3D model structure (Spahn et al, 2001) or the T. thermophilus 70S crystal structure (Voorhees et al, 2009), we determined corresponding regions in the T. thermophilus 16S rRNA and Haloarcula marismortui 23S rRNA. Using Pymol we then visualized the locations in the model structures (PDBs 1s1h, 1s1i and 2wdg). We used chimaera (http://www.cgl.ucsf.edu/chimera/) (Goddard et al, 2007) to manually dock the 40S model structure (Spahn et al, 2001) in cryo-EM reconstructions (emd_1211 and end_1212) according to the chimaera documentation. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Acknowledgements We thank Grzegorz Kudla for bioinformatics support and help with 4F, Bettina Bo¨ttcher for help with interpretation of cryo-EM images, Zhili Xu and Gloria Culver for providing the KsgA footprinting data ﬁgure, Simon Lebaron and Claudia Schneider for communicating unpublished results, Brigitte Pertschy for the pADH-HTP-Nob1 construct, the Edinburgh Gene Pool Sequencing Facility and the Swann Building kitchen staff. SG and DT designed the research. SG, EP and AG performed the research and analysed the data. SG and DT wrote the paper. This work was supported by the Welcome Trust, the BBSRC (BB/D019621/1), EMBO long-term fellowship (SG) and a Marie Curie EIF fellowship (SG). Conﬂict of interest The authors declare that they have no conﬂict of interest. of yeast 18S rRNA residues required for efﬁcient recruitment of initiator tRNA(Met) and AUG selection. 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