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Comparative Study
. 2019 May 7;47(8):4198-4210.
doi: 10.1093/nar/gkz106.

Differences in the path to exit the ribosome across the three domains of life

Khanh Dao Duc  1 , Sanjit S Batra  1 , Nicholas Bhattacharya  2 , Jamie H D Cate  3   4   5 , Yun S Song  1   6   7
Affiliations
Comparative Study

Differences in the path to exit the ribosome across the three domains of life

Khanh Dao Duc et al. Nucleic Acids Res. .

Abstract

The ribosome exit tunnel is an important structure involved in the regulation of translation and other essential functions such as protein folding. By comparing 20 recently obtained cryo-EM and X-ray crystallography structures of the ribosome from all three domains of life, we here characterize the key similarities and differences of the tunnel across species. We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure and sequence. We find that the tunnel is more conserved in the upper part close to the polypeptide transferase center, while in the lower part, it is substantially narrower in eukaryotes than in bacteria. Furthermore, we provide evidence for the existence of a second constriction site in eukaryotic exit tunnels. Overall, these results have several evolutionary and functional implications, which explain certain differences between eukaryotes and prokaryotes in their translation mechanisms. In particular, they suggest that major co-translational functions of bacterial tunnels were externalized in eukaryotes, while reducing the tunnel size provided some other advantages, such as facilitating the nascent chain elongation and enabling antibiotic resistance.

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Figures

Figure 1.
Figure 1.
Extraction of the ribosome exit tunnel coordinates. For a given structure of the ribosome large subunit (LSU) (from Schmidt et al. (97)), we first locate the PTC and then apply a tunnel search algorithm (26) to reconstruct the geometry of the exit tunnel (more details in ‘Materials and methods’ section). The inset in the right panel shows the exit tunnel with the PTC and the constriction site that separates the tunnel between its upper and lower parts.
Figure 2.
Figure 2.
Volume, length and average radius of the ribosome exit tunnel across different species. Horizontal bar plots represent the ordered volume (left), length (middle) and average radius (right) of the tunnels from our dataset. The volume and length are decomposed into two subparts, separated by the constriction site (see ‘Materials and methods’ section). Species are specified and colored by their respective domains (plus organelles, in yellow): bacteria (red), archaea (black), eukarya (blue).
Figure 3.
Figure 3.
Clustering of species obtained from pairwise comparison of the tunnel geometry. (A) For all our structures, we plot the tunnel radius as a function of the distance across the tunnel. These plots are used to compare the tunnel geometries. (B) Clustering obtained after applying our tunnel geometric distance metric to our dataset (for a definition of the metric and more details, see ‘Materials and methods’ section). The first main branch encompasses the bacterial ribosomes, highlighted in red, while the second contains eukarya (blue) and archaea (black) (scale bar: 0.2 Å). For the full clustering and phylogenetic trees obtained from 16S/18S rRNA sequences, see Supplementary Figure S2. (C) We divide for each couple of species their common domain after alignment in 4 quarters (see ‘Materials and methods’ section) and use the same metric to compute the distance in each of the subregions. The bar plots represent for each quarter the average and std of the geometric distance for subset of pairs made of 2 prokaryotes (red), 2 eukaryotes (blue), and 1 prokaryote and 1 eukaryote (violet).
Figure 4.
Figure 4.
The presence of a second constriction site in eukarya explains the geometric difference observed between bacterial and eukaryotic tunnels. (A) We show the constriction site region in E. coli (left) obtained from Fischer et al. (85), and H. Sapiens (right) obtained from Natchiar et al. (94). The structure is surrounded by ribosomal proteins uL4 and uL22. An extended arm in H. sapiens uL4 produces a second constriction site (see also Supplementary Figure S4). (B) The plots of the tunnel radius as a function of the tunnel distance shows a first trough associated with the constriction site (around position 30), common to E. coli and H. sapiens. A second trough appears around position 50. (C) We compare the distance between the troughs for bacteria (left box plot) and eukarya (right box plot) in our dataset. The interquartile range is indicated by the box, the median by a line inside, and upper and lower adjacent values by whiskers. (D) In left, we provide the same comparison as in (C) for the tunnel radii associated with the first and second troughs. In right, we compare the radius of the first and second troughs for each species of our dataset. The second trough radius is larger than the first one for all archaea (black dots) and bacteria (red dots). In contrast, this is only the case in eukarya for trypanosome species L. donovani and T. cruzi.
Figure 5.
Figure 5.
The replacement of uL23 by eL39 in eukarya affects the tunnel geometry. (A) The structures of the lower part of the tunnel in Escherichia coli (left) and Homo sapiens (right) show the replacement of ribosomal protein uL23 by eL39 in H. sapiens, which also covers a larger portion of the tunnel. (B) Upper plot shows a comparison of the distance covered by uL23 and eL39 in bacteria (right box plot) and eukarya (left box plot). Lower plot shows the same comparison for the average radius.
Figure 6.
Figure 6.
Association between geometric and sequence conservations of ribosomal rRNA. (A) A map of the secondary structure of the 23S rRNA in E. coli, colored by the distance from tunnel (see also Supplementary Figure S5). (B) For a given species and distance d, we look at all the rRNA nucleotides located within distance d from the tunnel, and we compute the frequency of conserved elements (24). We plot this frequency as a function of d for all the species of our dataset (see also Supplementary Figure S6). (C) We study the local conservation of rRNA nucleotides along the tunnel: Upon dividing the tunnel into regions of 15 Å along the centerline, we consider for each region all the rRNA nucleotides that are the closest and located within 25 Å, and we compute the associated number of conserved, domain-specific and universally conserved elements. We show here the resulting plots for E. coli (up) and H. sapiens (down) (for other species, see Supplementary Figure S6).
Figure 7.
Figure 7.
Conservation of sequence and positive charge of ribosomal protein uL22 at the tunnel. (A) We show the multiple sequence alignment of ribosomal protein uL22 close to the tunnel. Highlighted residues are the ones located within 10 Å from the tunnel. (B) We plot the sequence and charge conservation scores (see ‘Materials and methods’ section) along the sequence alignment. Continuous lines represent the signal averaged over a window of five sites. Larger highlighted region is the same as in (A). We also highlight a subregion of residues close to the tunnel, with a peak in charge or sequence conservation. (C) The associated structure of uL22 in H. sapiens, where residues in green and red correspond to the ones highlighted in (B). In particular, the region of high charge and sequence conservation is also in direct contact with the constriction sites. For the other ribosomal proteins associated with the tunnel, see Supplementary Figure S7.
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