the translation machinery. Yeast Dim1 can complement an E. coli ksgAD mutant (Lafontaine et al, 1994) and Dim1 cross- linking sites on the 18S rRNA included analogous positions (Supplementary Figure 5). We conclude that both the methyl transferase function of Dim1 and its interactions with the rRNA are conserved in evolution. Ribosome biogenesis in bacteria involves several different GTPases, which are proposed to act as ‘molecular switches’, Figure 5 Overview of known and predicted protein–protein inter- actions in pre-40S complexes. The interaction map depicts interac- tions between the various assembly factors and ribosomal proteins in pre-40S complexes. Black lines, physical interactions among proteins shown to be part of subcomplexes or interacting as recombinant proteins (Krogan et al, 2004; Vanrobays et al, 2004; Schafer et al, 2006; Tarassov et al, 2008; Lamanna and Karbstein, 2009). Dashed red lines, yeast two-hybrid interactions (Ito et al, 2001; Tone and Toh, 2002). Red lines, interactions from protein– fragment complementation assays (PCA), which detect proteins within 80 A˚ distance from each other (Tarassov et al, 2008). Dashed black lines, protein–protein interactions predicted from our CRAC data. Blue lines, physical interactions among bacterial homologues of the indicated proteins (Brodersen et al, 2002). Towards a structural framework for 40S assembly S Granneman et al regulating the stepwise assembly and maturation of RNA– protein subcomplexes (reviewed in Culver, 2001; Karbstein, 2007; Connolly and Culver, 2009). Two bacterial GTPases required for 16S rRNA processing (Era and RsgA/YjeQ) are genetically linked to KgsA (Inoue et al, 2006; Campbell and Brown, 2008). Cryo-EM microscopy studies revealed that some sites of Era interaction with the 16S rRNA are at positions analogous to the Tsr1-binding sites in yeast (H26, H28, H44, H45) (Sharma et al, 2005). Similarly, RsgA also contacts the 30 minor domain and GTP-bound RsgA causes structural rearrangements in H44 (Kimura et al, 2008). The overlap in RNA-binding sites observed for Dim1 and Tsr1 strongly suggest that they interact directly in pre-40S complexes. We predict that Tsr1 and Dim1 together fulﬁll functions in ribosome assembly that are equivalent to KsgA and Era/RsgA in bacteria. The Rio2 protein kinase is required for 20S–18S proces- sing, but its targets are unknown. In the 40S structure model, the Rio2-binding site is located close to Rps15, (Figure 3D) and pre-40S ribosomes that lack Rps15 fail to efﬁciently incorporate Rio2 and are not efﬁciently exported to the cytoplasm (Leger-Silvestre et al, 2004; Zemp et al, 2009). This suggests that these proteins interact directly. Human Rio2 kinase activity is required for release of hNob1, hLtv1 and hDim2 from pre-40S ribosomes (Zemp et al, 2009) and the binding sites for yeast Rio2, Ltv1 and Nob1 are closely located in the head domain. These sites are also close to Rps16 and Rps18 (Figure 3D). The bacterial Rps18 homo- logue (S13) is phosphorylated at serine and threonine residues (Soung et al, 2009) and this may also be the case for bacterial Rps16 (S9) (Traugh and Traut, 1972), suggesting Rps16 and Rps18 as potential Rio2 substrates. Pre-40S particles lack the prominent beak structure present in the mature subunit, implying large-scale structural reorga- nization during 40S maturation (Schafer et al, 2006). Two 40S synthesis factors, Enp1 and Ltv1, were implicated in this reorganization but their actual roles were unclear. We report that Enp1 directly binds sequences in H33 that will form the beak. Ltv1 binds sequences in H41, which are located close to the beak, but also binds H16, which is more distantly located in the shoulder region of the 40S particle. If Ltv1 binds both sequences simultaneously, it would need to span the head– shoulder gap—a distance of some 87 A˚ in the mature 40S subunit. Ltv1 is B53 kDa and, assuming a monomer with cylindrical shape and an average density of 0.73 cm3/g, an 87 A˚ long Ltv1 protein would have a diameter of B30 A˚ . Comparison of the cryo-EM maps for pre-40S and mature 40S Figure 4 At steady state the stem structure containing the D-cleavage site is highly ﬂexible and unbound by proteins. (A) Overview of the chemical foot-printing results on a secondary structure of ITS1, including the 50 end of the 18S rRNA, adopted from Yeh et al (1990). Red nucleotides indicate the location of the reverse transcriptase primer. Adenosines in H45 dimethylated by Dim1 in vivo are indicated with ‘-m’. Green circles indicate the nucleotide modiﬁed by DMS. Yellow and purple dots indicate the CMCT and 1M7-modiﬁed nucleotides, respectively. (B) The D-site region in puriﬁed 20S pre-rRNA is ﬂexible and unbound by proteins. DMS, CMCT and 1M7 modiﬁcation of RNA was performed at 301C on puriﬁed pre-40S complexes isolated using plasmid-expressed HTP-tagged Nob1 as bait. All chemicals primarily modiﬁed nucleotides in the D-site stem region, indicating it is ﬂexible or single stranded, whereas other regions predicted to be double stranded (i.e. H45 and Domain I) were largely protected from chemical modiﬁcation. Adenosines in H45 dimethylated by Dim1 in vivo are indicated with ‘-m’. (C–F) The D-site helix is highly ﬂexible and likely unbound by Nob1 in vivo. In vivo DMS was performed at 301C on total RNA puriﬁed from GAL::3HA-nob1 cells (E) grown in glucose for 8 h (C, lanes 1 and 2) or galactose (C, lanes 3 and 4). Depletion of Nob1 was conﬁrmed by western blotting (D) using an HRP-conjugated anti-HA antibody (Santa Cruz) and accumulation of 20S pre-rRNA was detected by ethidium bromide staining of total RNA in an agarose gel. Pgk1 antibodies (Santa Cruz) were used to conﬁrm loading of equal amounts proteins on each lane (D). After primer extension, radiolabelled cDNAs were resolved on 12% polyacrylamide/7 M urea gels and visualized by autoradiography. Adenosines in H45 dimethylated by Dim1 in vivo are indicated with ‘-m’. Quantiﬁcation of chemical probing data (F) was performed as described in the main text. & 2010 European Molecular Biology Organization The EMBO Journal VOL 29 | NO 12 | 2010 2033