Towards a structural framework for 40S assembly S Granneman et al revealed extra density the side of the head domain in pre-40S particles, close to the location predicted for Ltv1 (Figure 3B) (Schafer et al, 2006). The volume of this region would be in good agreement with the presence of a protein of B53 kDa (B Bo¨ ttcher, personal communication). To better facilitate the interpretation of the cryo-EM images, we manually docked the 40S structure model (1s1h) (Spahn et al, 2001) onto the 40S cryo-EM map (mesh model in Figure 3B) (Schafer et al, 2006) and overlaid this with the pre-40S cryo-EM map (transparent blue). This provided a reasonable estimation of location of ribosomal proteins and RNA structures in pre-40S pre-ribosomes. In the pre-40S EM map, the shoulder formed by H16 is absent or poorly deﬁned, however, the extra density appears to be located parallel to Rps3 and just above H16. We therefore predict that this density corresponds to Ltv1, although we cannot exclude the possibility that multiple copies of Ltv1 are present. The CRAC data revealed that Dim1, Tsr1 and Rio2 bind 18S rRNA regions that are important for the association of translation factors, tRNAs and 60S subunit joining. In the case of Dim1 and Tsr1, these are conserved to E. coli and in both bacterial and eukaryotic ribosomes are incompatible with binding to the mRNA, 60S subunit, initiator tRNA and translation factors. Dissociation of each of these proteins from the pre-40S particles would therefore be required for translation to commence. Pre-40S complexes were recently reported to associate with polysomes (Soudet et al, 2010), particularly after depletion of Nob1 or the Rio1 kinase. These conditions prevent 18S maturation, resulting in a very sub- stantial 20S pre-rRNA accumulation, which is readily visible by ethidium bromide staining of total RNA. We predict that not all of this large pool of accumulated pre-40S particles can be associated with the ribosome synthesis factors, and their absence may explain the ability of the defective pre-40S ribosomes to engage with the translation machinery (Soudet et al, 2010). Nob1 is the PIN-domain endonuclease that cleaves site D at the 30 end of 18S rRNA (Fatica et al, 2003; Pertschy et al, 2009). Unexpectedly, the major binding site identiﬁed for Nob1 was located in H40, distinct from the cleavage site. Binding to and cleavage of site D requires the PIN domain (Fatica et al, 2004; Lamanna and Karbstein, 2009; Pertschy et al, 2009). This indicates that H40 recognition involves a different region of Nob1, most likely the C-terminal, Zn-ribbon putative RNA-binding domain, potentially leaving the PIN domain free to recognize the cleavage site. Nob1 associates with 90S pre-ribosome early in 40S sub- unit synthesis pathway, but cleaves site D only in very late pre-40S particles after nuclear export (Fatica et al, 2003; Pertschy et al, 2009). Structure probing revealed that the region containing the D-cleavage site is readily accessible to chemical modiﬁcation within pre-ribosomes, both in vitro and in vivo. This indicates that it is largely unstructured and unbound by proteins, and should therefore be accessible to the nuclease. What then prevents Nob1 from cleaving site D in early pre-ribosomes? The pre-40S cross-linking data reported here and by Bohnsack et al (2009) identiﬁed four putative enzymes interacting with the decoding centre and with H44 (Tsr1, Rio2, Dim1 and Prp43), strongly implying that this region undergoes restructuring; as does the homo- logous region in bacteria. Notably, Dim1 also binds early, 2034 The EMBO Journal VOL 29 | NO 12 | 2010 nuclear pre-ribosomes, but methylates the 30 end of 18S rRNA much later in the pathway, after export to the cytoplasm. Restructuring might not only be essential for the correct folding of the 30 domain of the 18S rRNA, but also to allow cleavage at site D to occur. On the basis of 3D modelling, we speculate that only when the structure of 30 domain of 18S is close to its ﬁnal conformation, can Nob1 access and cleave site D. In 3D models, the Tsr1- and Dim1-binding sites are in close proximity to the 30 end of the 18S rRNA in the pre-40S and it is conceivable that both proteins could interfere with D-site cleavage by sterically hindering Nob1 binding to the cleavage site. We therefore predict that both RNA restructur- ing and protein remodelling steps in the 30 region of the 18S rRNA are necessary for Nob1-dependent cleavage at site D. Cryo-EM studies showed that binding of yeast translation initiation factors eIF1-eIF1A to the small subunit induces a conformational change that leads to a connection between the head and the shoulder, mediated by Rps3 and H16 (Passmore et al, 2007). This interaction stabilizes the head domain and prevents the ‘mRNA latch’ from forming prema- turely, making the mRNA-binding channel more accessible to the large cap-binding protein complex attached to the 50 end of the mRNA (Passmore et al, 2007). We propose that Ltv1 binding to both Rps3 and H16 locks the head into the pre- ribosome conformation, whereas Enp1 binding to Rps3 and H33 directly prevents formation of the beak structure (Figure 3C). The 40S subunit is characterized by structural changes during the translation cycle, and it appears that large-scale structural changes also feature it its maturation. Materials and methods Yeast strains and media S. cerevisiae strain BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0) was used as the parental strain (Brachmann et al, 1998). The HTP carboxyl-tagged strains (Supplementary Table 3) were generated by PCR as described (Rigaut et al, 1999) using oligonucleotides listed in Supplmentary Table 2. Strains were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) or YPG/R (YP with 2% galactose and 2% rafﬁnose) at 301C. Cross-linking and analyses of cDNA (CRAC) In vivo CRAC, western and northern blot analyses were performed as described earlier (Granneman et al, 2009) with the following modiﬁcations: for the Ltv1 and Tsr1 CRAC experiments TEV eluates were incubated with lower amounts of RNase (0.1–0.5 units of RNaceIT (Stratagene), depending on the batch used) for 5 min at 371C. For cloning the 50 Solexa and miRCat-33 linkers were used (Integrated DNA technologies, UK) (Granneman et al, 2009). Sequence analysis Sanger sequencing of cDNAs was performed as described (Granne- man et al, 2009). Sequences were analysed with Blast (http:// blast.ncbi.nlm.nih.gov). The sequences (inserts) located between pairs of linkers were extracted and analysed for homology with yeast RNAs. The choice of blast parameters did not qualitatively affect the results; we typically used word size, 8; expectation value, 0.1; other parameters were set to default. We then computed, for each position along the sequence of interest, the number of inserts covering that position. The Blast output was used to generate histograms shown in Figure 1 and Supplementary Figure 3. To map the substitutions and deletions and generate sequence alignments, fasta ﬁles containing the insert were aligned to the rDNA reference sequence using Novoalign 2.04 (http://www.novocraft.com) as described earlier (Granneman et al, 2009). The Novoalign output was used to generate the multiple sequence alignments shown in Supplementary data online. A set of Awk and Perl scripts for automated sequence analysis in Windows, Mac or Linux environ- ments is available on request. & 2010 European Molecular Biology Organization