Towards a structural framework for 40S assembly S Granneman et al Figure 2 Locations of protein–RNA interaction sites in the 18S rRNA secondary structure. (A) Overview of the yeast 18S rRNA secondary structure (obtained from http://www.rna.ccbb.utexas.edu/). The stem including the D-cleavage site was added separately. The 18S rRNA domains are indicated as dashed boxes. Functionally, important elements (central pseudoknot, decoding centre, P-site tRNA interactions, D-cleavage site (coloured red)) are marked with circles. (B) Overview of RNA-binding sites for pre-40S-associated proteins. Speciﬁc nucleotides cross-linked to proteins are indicated as coloured circles. For cases where no or few mutations were found in cross-linked RNAs (Tsr1 H26 and H27, Dim1 H28 and the Enp1-binding site in H34), the binding sites are indicated by dashed lines. Dashed boxes indicate predicted ribosomal protein (Rps) interaction sites on the 18S rRNA, based on the locations of homologous T. thermophilus ribosomal proteins in the 30S crystal structure, the yeast 40S structure model and various other studies performed on ribosomal proteins in yeast (Spahn et al, 2001; Brodersen et al, 2002; Antunez de Mayolo and Woolford, 2003; Dresios et al, 2005). Helix numbering was adopted from Brodersen et al (2002). RNA-binding sites for the RNA helicase Prp43 (Bohnsack et al, 2009) are also included. with Tsr1 was 41 nt, against an overall average of 28 nt for other pre-40S proteins. This indicates that this region is relatively resistant to RNase digestion, suggesting a stable structure. Shorter fragments were also identiﬁed in helices 26, 27 and in the region spanning helices 2 and 28, with the latter overlapping the Dim1-binding site (Figure 2B). Among the Tsr1 reads derived from the H28 region, 25% contained mutations in a single-stranded sequence located between H2 and H28 (nt 1143–1146), precisely deﬁning the Tsr1-binding site in this region. Notably, these mutations were not found in the Dim1- binding site in the H28 region (Supplementary Table 4). Twenty ﬁve per cent of Tsr1 hits mapped to the 30 minor domain of 18S rRNA, in H44 and H45 (Figure 1A), close to the cleavage site at the 30 end of the 18S rRNA (site D) and partially overlapping the major Dim1-binding site (Figure 2A and B). Among the H45 hits, 44% contained mutations (nt 1765–1771, Figure 2B), identifying the direct binding site. Tsr1 binding near site D is consistent with a proposed direct role in 18S rRNA processing (Gelperin et al, 2001). In the crystal structure of the 30S Thermus thermophilus ribosomal subunit, H26 and H27 loop around H45 in the vicinity of the 30 end of the 16S rRNA (see Figure 3E). In addition, the hairpin loop of H27 makes extensive minor & 2010 European Molecular Biology Organization groove contacts with H44 just below the decoding centre (see Figure 3E) (Clemons et al, 1999; Voorhees et al, 2009). Tsr1 could therefore bind simultaneously with the H27–28 and H44–45 regions. A lower frequency of Tsr1 cross-linking was recovered with H34 (Figure 1A), close to the Rps5-binding site in regions involved in P-site tRNA interactions (Figure 2A) (Spahn et al, 2001). The Tsr1 hits in this region are not the same as the negative control, indicating that this interaction is speciﬁc. We conclude that both Dim1 and Tsr1 bind to sequences in the 18S rRNA that includes the decoding centre and regions required for tRNA association with the ribosome. These binding sites are incompatible with the formation of a trans- lation initiation complex. For example, the Dim1-binding sites in H44 overlap with translation initiation factor eIF1 (Lomakin et al, 2003; Passmore et al, 2007), whereas binding of Tsr1 and Dim1 to H28 would interfere with association of the initiator tRNA (Dong et al, 2008). Thus, Tsr1 and Dim1 are likely to prevent pre-40S complexes from participating in translation. In addition, the Tsr1- and Dim1-binding sites enclose the 30 end of the 18S rRNA (Figure 3E), suggesting that their association would impede access of the Nob1 endonuclease to site D (see below). The EMBO Journal VOL 29 | NO 12 | 2010 2029