The molecular models of the S. cerevisiae and T. aestivum 80S ribosomes were used to analyze the architecture of the eukaryotic ribosomal tunnel exit site. The tunnel exit site consists of rRNA helices H6, H7, H24, H47, H50, H53 and H59 and the r-proteins L4e (rpL4), L19e (rpL19), L22p (rpL17) L23p (rpL25), L24p (rpL26), L29p (rpL35), L31e (rpL31) and L39e (rpL39) (Figure 10.3.1 A, B). The overall architecture of the tunnel exit site is very similar compared to bacteria, however, signicant dierences in H7, H24 and H59 are present. In both, S. cerevisiae and T. aestivum, H7 adopts a newly and unidentied kink-turn fold comparable to E. coli. As in bacteria H7 makes a pseudoknot interaction with the adjacent H6. The interaction between the loops of H6 and H7 is stabilized by the eukaryote-specic r-protein L39e which replaces the loop of the bacterial counterpart L23. Another dierence is observed for the H24 loop region. In bacteria the loop of H24 is a pentaloop. In contrast, the loop of H24 in fungal and plant ribosomes is a GNRA-tetraloop, which is clearly visible in the cryo-EM densities. Although the loop motif and structure of the loop changes, the stacking interaction with the loop of H47 is conserved. The expansion segmentES24Lis the extension of H59. The loop ofES24Lis a tetraloop of unknown structure in S. cerevisiae and a pentaloop in T. aestivum, respectively.
10. Molecular interpretation of functional sites of the eukaryotic 80S ribosome
Figure 10.3.1.:Visualization of the eukaryotic ribosomal exit tunnel. (A) Bottom view of the S. cere- visiae cryo-EM map at 6.1 Å with highlighted rRNA (blue) and r-proteins (yellow, orange and brown, respectively). The asterisk indicates the tunnel exit. (B) Same as in (A), showing the molecular models for rRNA helices (blue) H6, H7, H24, H50 and H59 as well as r-proteins L4e, L29p and L19e (in yellow), L22p, L23p and L24p (in orange) and L31e and L39e (in brown). The r-proteins are named by their family name. The gures were taken from [141].
10.3.1. Interaction of the protein-conducting channel and the tunnel exit site One of the main interaction partners with the ribosomal exit site is the the protein-conducting channel (PCC) studied in this work[141]. The PCC of the canonical secretory pathway is formed in all cells by the Sec61/SecY complex. The PCC enables post- and co-translational translocation of secretory proteins into the membrane of the endoplasmic reticulum in eukaryotes and plasma membrane of bacteria [190, 191].
On the basis of the molecular model it was possible to characterize the interactions between eukaryotic actively translating ribosomes and the PCC[141].
Sample 80S ribosomes of T. aestivum and S. cerevisiae were programmed with a truncated mRNA coding for the rst 120 amino acids of DPAP-B (DP120) carrying a signal sequence for co-translational protein translocation. These RNCs were in vitro reconstituted with puried Ssh1p or mammalian Sec61 (mSec61) complexes embedded in a detergent micelle. Cryo-EM structures of the T. aestivum DP120-RNC-mSec61 complex and the S. cerevisiae DP120-RNC- Ssh1p complex were obtained at 6.5 Å and 6.1 Å, respectively. At this resolution, the extended NC could be visualized from the PTC to the tunnel exit site for the rst time. Moreover, for the mammalian Sec61 complex most of the transmembrane helices could be resolved and assigned. On this basis, a single Sec61 heterotrimer surrounded by a mixed detergent/lipid micelle was identied. This could also be shown for the Ssh1p and allowed the interpretation of the ribosome- PCC interaction on a molecular level. The S. cerevisiae RNC-Ssh1 complex was prepared by Dr. Thomas Becker (RNCs) and Dr. Elisabeth Menden (Ssh1p) and the T. aestivum RNC-mSec61 complex was prepared by Dr. Shashi Bhushan (RNCs) and Dr. Soledad Funes (mSec61). The processing was done by Dr. Thomas Becker. The model of the PCC was constructed by Dr. Thomas Becker. The models for the r-proteins were constructed by J.-P. Armache.
10. Molecular interpretation of functional sites of the eukaryotic 80S ribosome
Interactions of the PCC with the ribosome Both the Ssh1p and mSec61 monomers use the universal ribosomal adapter site as a main contact. The main connections are established by the cytoplasmic loops L6 and L8 (Figure 10.3.2 A). In Ssh1p, loop L6 directly interacts with the loop of H7 (backbone). The loop L8 interacts with the loop of H6 (backbone) and H50 (minor groove and backbone). L8 is also stabilized by r-proteins L23p (rpL25) and L29p (rpL35). Two additional connections are established between i) the loop of the eukaryote-specic RNA expansion segment ES24L (H59), which interacts with the N-terminus of Ssh1p, and ii) H24 together with L24p (rpL26) stabilizing the C-terminus of Ssh1p (Figure 10.3.2 A, B).
Figure 10.3.2.:Interaction of Ssh1p with the ribosome. (A and B) S. cerevisiae 80S ribosome (Thumb- nail insets). (A) Molecular models for rRNA (blue) and r-proteins L23p (orange), L29p (yellow) and L39e (brown). The cytosolic half of the Ssh1p and Sss1 model is shown in red and magenta, respectively. The positions of the conserved R278 and R411 are indicated (green). (B) Bottom view. The red line indicates the shape of the Ssh1p. The density for the nascent chain in the tunnel exit site is shown in green. The gures were taken from [141].
10. Molecular interpretation of functional sites of the eukaryotic 80S ribosome
The connections between the 80S ribosome and the mammalian Sec61 complex are similar to those of Ssh1p. Hereby, loop L6 interacts with the loop of H6, H50, L29p (rpL35) and L39e (rpL39). Loop L8 contacts L23p (rpL25), H50 and also H53. As in the Ssh1p homolog the N-terminus of mSec61 interacts with the loop of rRNA expansion segment ES24L, while the C-terminus is stabilized by H24 together with L24p (rpL26) (Figure 10.3.3 A).
Figure 10.3.3.:Interaction of mammalian Sec61 (mSec61) with the 80S ribosome. (A) Side view of the T . aestivum 80S ribosome bound to the mSec61 complex. Molecular models for rRNA (blue) and r-proteins L23p (yellow) and L39e (brown). The mSec61 complex is shown in red. Loop L6 interacts with H6, H50, L29p and L39e. Loop L8 interacts with H50 and L23p. The gure was taken from [141].