Towards a structural framework for 40S assembly S Granneman et al Figure 1 Overview of CRAC results. Shown are the results from independent CRAC experiments performed on pre-40S-associated proteins (A). Results from untagged strains are shown in (B). Sequences were aligned to the rDNA reference sequence using blast and plotted using gnuplot. The locations of mature rRNA sequences, spacers and cleavage site are indicated below the x axis. The y axis shows the total number of times each nucleotide within an RNA fragment was mapped to the rDNA sequence. The location of the peaks in the secondary structure of the rRNA (see Figure 2A, B) is indicated with helix (H) numbers. The asterisks indicate frequent contaminants. and Rps29 (Figures 2B and 3D) (Spahn et al, 2001; Brodersen et al, 2002). The Rio2 cross-linking sites are located in the cleft that, in the 80S ribosome, is occupied by the P-site tRNA and the C-terminal domains of Rps16 and Rps18 (Spahn et al, 2001). This makes it very likely that the association of Rio2 with the pre-40S particles is incompatible with tRNA binding. Dim1 and the putative GTP-binding protein Tsr1 interact with the central pseudoknot and the decoding centre Dim1 dimethylates two adenosines in the loop of H45 (in- dicated as ‘-m’ in Figure 2B) (Lafontaine et al, 1994) and the methylation sites were recovered in several sequence reads. However, a major Dim1 cross-linking site was located in the adjacent H44 region (nt 1753–1794) (Figures 1 and 2B; Supplementary Table 4). A second major peak covered se- quences over H2 and the adjacent H28 region (nt 1137–1156). H2 base pairs with the 50 end of 18S rRNA to form the central pseudoknot. Strains lacking Dim1 are unable to cleave site A1 2028 The EMBO Journal VOL 29 | NO 12 | 2010 at the 50 end of 18S rRNA (Lafontaine et al, 1995), and we predict this requirement reﬂects its interaction with the central pseudoknot. H28 is required for recruitment of the initiator methionine tRNA (Dong et al, 2008) and Dim1 binding here would be incompatible with tRNAiMet associa- tion. Cross-linking to H11, near the Rps11-binding site (Dresios et al, 2005), was also reproducibly observed but with fewer hits (Figures 1A, 2B and 3E). Tsr1 is required for cleavage of the 20S pre-rRNA at site D to generate 18S rRNA (Gelperin et al, 2001) and has a region related to the GTPase domain of elongation factor Tu, although GTP binding and hydrolysis have not yet been reported. The major binding site for Tsr1 was located in the central domain of the 18S rRNA over H19 and H26–29 (Figures 1A and 2B; Supplementary Table 9). These se- quences form a large domain within the platform and neck regions of 40S subunits (Spahn et al, 2001) (Figure 2B). The average read-length for H19–H26–29 sequences associated & 2010 European Molecular Biology Organization