High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

Abstract

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.

Fig. 1

High-resolution cryo-EM structure of the T. brucei ribosome. The density map was filtered with a locally varying bandpass, according to the local resolution measurements (see Methods and figure S2A). In all following panels, largest rRNA expansion segments (ESs) are rendered in different colors. (a) The T. brucei ribosome viewed from the front (P-stalk and beak side), (b) viewed from the back (L1-stalk and platform side), (c) solvent side of the small subunit (SSU) and (d) solvent side of the large subunit (LSU). KSD = kinetoplastid-specific domain, hd = head, bk = beak and cp = central protuberance.

Fig. 2

Atomic model of the T. brucei ribosome. (a) SSU and (b) LSU: atomic models with the ESs colored and annotated similarly as in Fig. 1. (c) SSU and (d) LSU: atomic models in surface presentations. Gray regions indicate the location of conserved common core elements of all ribosomes, prokaryotic and eukaryotic. Yellow regions highlight eukaryotic-specific conserved elements, including those for trypanosomes. Red regions indicate trypanosome-specific elements, nonexistent in other 80S ribosomes.

Fig. 3

Comparison between T. brucei and yeast ribosomes. View of rRNA expansion segments (ES) at the back of the T. brucei (a) and yeast (b) ribosomes. T. brucei-specific intersubunit bridges are indicated by double arrows in (a). srRNA-I cleavage site in T. brucei and its counterpart region in yeast are blown up at the bottom and highlighted by red frames. Black circles highlight S6e interaction with ESs 3S & 6S in both ribosomes. Red spheres in (a) denote srRNA I and IV cleaved ends. Tb-spfc. ext. = T. brucei-specific extension.

Fig. 4

T. brucei srRNAs and protein extensions. (a) Left and middle, srRNAs II to IV and KSD cryo-EM density segmentations along with their atomic models compared to yeast domain VI (right). Thick Green arrow highlights the kissing-loop interaction. Black circle surrounds ES39L in yeast ribosome. (b) Ribosomal proteins presenting specific extensions in T. brucei (cyan ribbon) with zooms on the interactions of their extensions with the surrounding rRNA. (c) Left, cryo-EM-derived model of the eIF3-binding site (recreated according to ref. 21). Right, segmented map of the SSU, from T. brucei ribosome, filtered to 20Å. KSD = kinetoplastid-specific domain.