primarily binds over the loop of H40, with only transient interactions at cleavage site D. The PIN domain is characterized by three Asp residues that coordinate a divalent metal ion. Previous analyses showed that mutation of one of these, D15N inhibits site D cleavage in vivo and in vitro (Fatica et al, 2003; Pertschy et al, 2009). This mutation does not disrupt Nob1 binding to the 20S pre- rRNA in vivo and CRAC analysis on HTP-Nob1 D15N showed that the active site mutation did not dramatically affect association with the 20S pre-rRNA or cross-linking to H40 in vivo (Supplementary Figure 4B and C), indicating that the nuclease activity is not required for binding to H40. The HTP- Nob1 D15N mutant is dominant negative and causes accu- mulation of 20S pre-rRNA (Supplementary Figure 4C), pre- sumably because the mutant protein binds to pre-ribosomes but does not catalyse cleavage. However, the D15N mutant also failed to show signiﬁcant cross-linking at the D-site region (Supplementary Figure 4B). Nob1-directed cleavage at site D is a late step in subunit maturation, so the structure of the cytoplasmic pre-40S particle is probably close to the ﬁnal conformation. In the 3D model of the Saccharomyces cerevisiae 40S subunit (Spahn et al, 2001), the Nob1 cross-linking site lies B33 A˚ away from the 30 end of the 18S rRNA, equivalent to a stretch of about 10 nucleotides (Figure 3F). However, the model structure lacks the last 8 nt of 18S rRNA, so the actual distance to site D may be less. The dimensions of the dimer of the hSmg6 PIN domain are 36–71–181 A˚ (Glavan et al, 2006). Nob1 is predicted to have similar dimensions and was reported to bind RNA in vitro as a tetramer (Lamanna and Karbstein, 2009). We therefore predict that Nob1 bound to H40 could simultaneously interact with site D. The CRAC data indicated that the majority of pre-40S ribosomes do not have Nob1 bound at site D. To conﬁrm that this does not simply reﬂect inefﬁcient cross-linking because of RNA structure or other features, dimethyl sulfate (DMS) chemical foot-printing experiments were performed in vivo on total pre-ribosomes and in vitro on pre-40S com- plexes (Figure 4). DMS methylates adenines in single stranded or ﬂexible RNA unbound by proteins. The pre-40S particles were puriﬁed by precipitation of plasmid expressed HTP-tagged Nob1, which substantially enriched for 20S pre-rRNA (Supplementary Figure 4C). Primer extension analysis of pre-ribosomes modiﬁed with DMS in vitro (Figure 4B, lane 4) or in vivo (Figure 4C) revealed an almost identical pattern of primer extension stops in the region containing the D cleavage site. The high degree of methylation in the D-site stem indicates that this region is ﬂexible, consistent with previous results (Lamanna and Karbstein, 2009). In contrast, most adenosines in helix 45 and domain I were largely protected from chemical modiﬁca- tion, consistent with a stem structure. The DMS probing results were conﬁrmed by in vitro modiﬁcation of the 20S rRNA in puriﬁed pre-40S complexes with 1-methyl-7-nitro- isatoic anhydride (1M7; Figure 4B, lane 2), which modiﬁed 20-OH residues in single-stranded or ﬂexible regions (Mortimer and Weeks, 2007), and 1-cyclohexyl-(2-morpholi- noethyl)carbodiimide metho-p-toluene sulfonate (CMCT) (Figure 4B, lane 3), which modiﬁes unpaired uridines not bound by proteins. Collectively, the cleavages observed are consistent with a largely open structure and indicate that, at least in a subset of particles, Nob1 does not contact the D-site & 2010 European Molecular Biology Organization Towards a structural framework for 40S assembly S Granneman et al region in puriﬁed pre-40S complexes. To substantiate these results we probed the D-site region in Nob1-depleted cells. A glucose repressible Nob1 strain (GAL-3HAHnob1) was grown to exponential phase, shifted to glucose containing medium and grown for 8 h at 301C. Nob1 protein levels were signiﬁcantly reduced after 8 h in glucose containing medium (Figure 4D) and a substantial accumulation of 20S was detected in these cells (Figure 4E). To quantify the chemical probing data (Figure 4C), signal intensities for each band were normalized to remove differences between lanes. This revealed no signiﬁcant changes in the intensity or pattern of modiﬁcations near the D-site region in Nob1-depleted cells (Figure 4F). We conclude that in pre-40S pre-ribosomes, site D region is very ﬂexible and at steady-state Nob1 is likely not stably associated with the D-cleavage site. The region con- taining the D-site is normally drawn as a stem structure (Yeh et al, 1990) (Figure 4A); however, these data and recent in vitro and in vivo analyses (Lamanna and Karbstein, 2009) do not support this stem structure. These observations suggest that alterations in pre-ribo- some structure facilitate site D cleavage. Release of Tsr1 and Dim1 may be required for access of Nob1 to the 30 minor domain. Alternatively, the conformational change in the head domain might bring Nob1 into the correct position for cleavage at site D, and these possibilities are by no means mutually exclusive. Discussion Here, we have presented a protein–RNA interaction map for the late pre-40S ribosomes, providing insights into their architecture and maturation. Importantly, we found a good correlation between the CRAC data and previous protein– protein interaction and biochemical data, underlying the reliability of the method. We can now signiﬁcantly extend the interaction maps as shown in Figure 5. No crystal structures are available for eukaryotic ribo- somes, and the best available structure model for the yeast ribosome was generated using cryo-EM reconstructions and homology modelling (Spahn et al, 2001). To relate the binding sites identiﬁed in the primary sequence to the 3D structure, we ﬁrst identiﬁed the corresponding sequence in the archaeal rRNA and its position in the crystal structure, which was used to locate the binding site in the yeast structure model. A striking ﬁnding was that cross-linking sites for ﬁve of the late-acting 40S synthesis factors Rio2, Tsr1, Dim1, Nob1 (this work) and Prp43 (Bohnsack et al, 2009) are located in close proximity to functionally important sequence elements in the 30 region of the 18S rRNA. Intriguingly, the rRNA-binding sites appear to be located in proximity to ribosomal proteins previously shown to be required for D-site cleavage and/or efﬁcient nuclear export of the pre-40S complex (Rps2, Rps3, Rps15 and Rps20, Rps0 and the C-terminus of Rps14) (Tabb- Massey et al, 2003; Jakovljevic et al, 2004; Leger-Silvestre et al, 2004; Ferreira-Cerca et al, 2005). This could reﬂect a general mechanism by which assembly factors prevent stable binding of ribosomal proteins before rRNA maturation steps are completed, as has been proposed for the association of Enp1 and Ltv1 with Rps3. Notably, the binding sites identiﬁed predominately lie in the mature 18S rRNA region, rather than in the trans- cribed spacers, and are far more common over evolutionarily The EMBO Journal VOL 29 | NO 12 | 2010 2031