Towards a structural framework for 40S assembly S Granneman et al Figure 3 Modelling of protein–RNA interaction sites into 3D structures. (A) Modelling of protein–RNA-binding sites in the 40S structure model (pdb 1s1h; Spahn et al, 2001). Shown is the model structure of the 18S rRNA. Characteristic structural features are indicated as head, beak, neck, shoulder and platform. Enp1- and Ltv1-binding sites are located near the beak formed by H33. Shown in wheat colour is the 18S rRNA tertiary structure superimposed with ribbon structure models for Rps3 (light blue), Rps9 (green) and Rps20 (yellow). H16 and H41A that interact with Ltv1 are shown in black. Enp1 cross-linking sites in H33 are depicted as red dots. (B) Ltv1 may correspond to the extra density near the head and the shoulder in the head domain. The docked molecular model of the yeast 40S subunit is shown for the 40S cryo-EM map (cage) superimposed on the pre-40S cryo-EM map (transparent blue). Helices 16 and 41 surfaces (Ltv1-binding sites) are indicated in yellow, Enp1-binding site surface is indicated in red, Rps3 is indicated in blue and Rps16 is indicated in orange. (C) Model for beak formation in pre- 40S complexes. On the schematic representation of pre-40S cryo-EM reconstruction images (Schafer et al, 2006) the predicted location of Rps3 is indicated in blue. In the model, Hrr25-dependent phosphorylation of Rps3 and subsequent dephosphorylation trigger a structural change leading to repositioning of Rps3, releasing of Enp1 and Ltv1 and beak formation (Schafer et al, 2006). Included are the predicted locations of Ltv1 (spanning the head and shoulder domain) and Enp1 (in the centre next to Rps3) in the pre-40S pre-ribosome. (D) Rio2, Ltv1 and Nob1- binding sites cluster around Rps16. The surface density of Rps15 and Rps16 in the head domain of the small subunit is shown in green and orange, respectively. The cross-linking sites for Ltv1 (black), Nob1 (red) and Rio2 (blue) are indicated as dots. (E) Tsr1 and Dim1 interact with rRNA near the decoding centre. The Tsr1- and Dim1-binding sites were modelled in the T. thermophilus 30S crystal structure with bound mRNA (PDB 2wgd) (Voorhees et al, 2009). Shown are helices 44, 45 and the RNA segments containing the Tsr1- and Dim1-binding sites (helices 11,26,27,28). The decoding centre is roughly marked with a dashed circle. (F) Nob1 binds the head domain in close proximity to the 30 end of the 18S rRNA. Shown is the tertiary structure model of the 18S rRNA. Nob1 and Prp43 cross-linking sites are indicated as coloured dots. Helix 44 is indicated in black. The arrows indicate the distance (33 A˚ ) between the Nob1 cross-linking site and the modelled 30-end of the 18S rRNA. The region surrounding cleavage site D is ﬂexible and not stably associated with Nob1 in pre-40S ribosomes Nob1 is a PIN-domain endonuclease that cleaves site D at the 30 end of mature 18S rRNA in vivo and in vitro (Fatica et al, 2003; Pertschy et al, 2009). Unusually for a nuclease, Nob1 is stably associated with its substrate. Nob1 interacts with early pre-ribosomal complexes (90S pre-ribosomes) in the nucleo- lus but remains associated with the pre-40S particles during their export to the cytoplasm, where site D cleavage takes place. This raises the question of what prevents premature, nuclear pre-rRNA cleavage by Nob1? In CRAC analyses, the most frequent site of Nob1 cross- linking was not at site D, but over helix 40 in the head domain, over 300 nt upstream (Figures 2B and 3F). Of these sequences, 84% contained micro deletions in the terminal loop of H40, precisely identifying the cross-linked nucleotides (Figure 2B, nt 1396–1398). In addition, a few sequences were mapped to H28 (Figure 1A). This indicates that Nob1 2030 The EMBO Journal VOL 29 | NO 12 | 2010 & 2010 European Molecular Biology Organization