Small Subunit Proteins

Figure 22. Localization of Eukaryote-specific r proteins. Cryo-EM maps of the T. aestivum 40S subunit (A) and (B) molecular models of r proteins, with newly identified r proteins colored distinctly.

Bacterial ribosomal small subunit contains up to 23 proteins, of which 8 exist only in this domain. Eukaryotic one in turn comprises 33 proteins, of which 13 are Archaea/Eukarya- specific and 5 are found only in the latter. We managed to assign the location of twelve small

3 Results subunit proteins, of which 2 are Eukarya-only and the rest exist both in Eukaryotes and Archaea.

The assignment of location of S19e (rpS19) to the head of the 40S subunit was based on the order of assembly of precursor particles (Ferreira-Cerca et al., 2007). Additional data, like the structural information provided by X-ray and NMR, as well as immuno-EM and cross-links (Figure 23, Figure 24), contributed to localization of the other 11 small subunit proteins.

Figure 23. Model of the spatial arrangement of proteins within the small ribosomal subunit based on cross- linking and immuno-EM data (adapted from (Gross et al., 1983)). (A-D) represent different views of the SSU.

Figure 24. Four different views of the 405 subunit model indicating the location of ribosomal proteins, based on immuno-electron microscopy studies (adapted from Bommer et al., 1991).

3 Results

3.3.1.1 Head of the Small Subunit

The position of RACK1 is in agreement with localization of this protein to the back of the head of the small subunit in fungi and mammalian ribosomes (Chandramouli et al., 2008; Sengupta et al., 2004), however we do not observe any evidence for asymmetry of the RACK1 β-propellers (Figure 7B), as suggested previously for the canine 80S ribosome (Chandramouli et al., 2008). Moreover, we can assign the unidentified protein interaction partner of RACK1 as being the Eukaryote-specific C-terminal extension of r protein S2p. The localization of S19e to the head of the 40S subunit is consistent with biochemical data of assembly precursor particles formed in vivo (Ferreira-Cerca et al., 2007). In addition, an X- ray structure of S19e from P. abyssi (Gregory et al., 2007) revealed a unique fold, which immediately pointed to the exact location of this protein.

The loops of S19e located between α1 and α2 as well as α4 and α5 are disordered in the crystal structure (Gregory et al., 2007), but become ordered upon ribosome binding where they interact with the variable region of helix41 (h41) of the 18S rRNA. Mutations in S19e found in Diamond-Blackfan anemia (DBA) patients are clustered around α3 (Gregory et al., 2007), which is also seen to interact with h41 in the T. aestivum and S. cerevisiae 80S models. DBA is an inherited bone marrow failure syndrome that results from defects in ribosomal assembly (Freed et al., 2010).

3.3.1.1.1 mRNA Entry

The interactions between the beak and h18 of the small subunit form the mRNA entry tunnel latch. The beak is described as a variable region (Armache et al., 2010a) and has a substantially different structure to that of the Prokaryotes. It is also a place where a new Eukaryote-specific protein, S17e (rpS17) was placed tentatively.

3.3.1.1.2 mRNA Exit

Three Eukaryote-specific r proteins, S21e, S26e, and S28e, were identified at the mRNA exit site between the platform and head of 40S subunit (see Figure 25A). Both S26e and S28e have been cross-linked from positions (−6 and −7⁄ − 10, respectively) in the 5’ untranslated region (UTR) of mRNA (Pisarev et al., 2008). The equivalent region of bacterial 30S subunits is occupied by bacterial-specific r proteins S6, S8, as well as S21 in E. coli (Schuwirth et al.,

3 Results 2005; Wimberly et al., 2000) (see Figure 25B). These differences may reflect the distinct elements found in the 5’ UTRs of bacterial and eukaryotic mRNAs, as well as the divergence in the translation initiation phase (Sonenberg and Hinnebusch, 2009).

Figure 25. (A) Small 40S subunit with newly modeled r proteins S21e, S26e, and S28e colored distinctly (thumbnail, Left; zoom, Right). (B) Comparative view of the bacterial 30S subunit with bacterial-specific S18p shown in green (Selmer et al., 2006). In A and B, the P-tRNA (blue) and mRNA (orange) are shown for reference.

3.3.1.1.3 Decoding Center

Although the active sites of the ribosome - the decoding site on the small subunit and the site of peptide-bond formation on the large subunit - are composed largely of rRNA, they are not completely devoid of r proteins (see Figure 17 and Figure 26). Compared with bacterial 30S subunits, eukaryotic 40S subunits contain two additional r proteins, S25e and S30e, with extensions that reach into the decoding and tRNA binding sites (see Figure 26A and B). Consistent with this localization, S30e was cross-linked to the 4-thiouridine containing UGA stop codon of mRNA positioned at the A-site (Bulygin et al., 2005).

Figure 26. (A)Small 40S subunit with newly modeled r proteins S30e and S25e (red) and Eukaryote-specific extension of S4p (green) highlighted (thumbnail, Left; zoom, Right). (B) Comparative view of the bacterial 30S subunit decoding site (Jenner et al., 2010; Selmer et al., 2006). In A and B, the anticodon-stemloops of A-, P- and E-tRNAs (blue) and mRNA (orange) are shown for reference.

3 Results

3.3.1.3 Small Subunit Body

Four small subunit proteins were localized on the body of the 40S subunit, namely S4e, S7e, S24e and S27e. Information about their position was derived from the immuno-electron microscopy studies (for all of them, see Figure 24), as well as the structural data available for S4e, S24e (Choesmel et al., 2008) and S27e (Herve du Penhoat et al., 2004). A general position of those proteins was established and the available structures were tentatively fit into the density. This resulted in points of references for localization of S7e into the only remaining density in the region pointed out by immuno-EM.