Structural insights into species-specific features of the ribosome from the human pathogen Mycobacterium tuberculosis

Abstract

Ribosomes from Mycobacterium tuberculosis (Mtb) possess species-specific ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein). Here, we present the near-atomic structures of the Mtb 50S ribosomal subunit and the complete Mtb 70S ribosome, solved by cryo-electron microscopy. Upon joining of the large and small ribosomal subunits, a 100-nt long expansion segment of the Mtb 23S rRNA, named H54a or the 'handle', switches interactions from with rRNA helix H68 and rProtein uL2 to with rProtein bS6, forming a new intersubunit bridge 'B9'. In Mtb 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. These results provide a structural basis for studying translation in Mtb as well as developing new tuberculosis drugs.

Figure 1.

Cryo-EM structures of the Mtb 50S and 70S ribosomes. (A) Secondary structure of the 5′ half of the Mtb 23S, showing the positions of the Mtb expansion segments (plum color) within the 23S rRNA. (B) The model of a helix fragment of the 23S rRNA (residue 818–822 and residue 898–902) fits into the density from the Mtb 50S, showing individual RNA bases. (C) The model of bL35 (residues 6–31) fits into the density from the Mtb 70S, showing bulky protein side chains. (D) Overall structures of the Mtb 50S (top row) and 70S (bottom row) ribosomes viewing from the subunit interface (left column) and the L7/L12 stalk base (right column), respectively. Structural landmarks of the bacterial ribosome are labeled. Color schemes are dodger blue for LSU rProteins, light blue for 23S, plum for 23S rRNA expansion segments, green for 5S, gold for SSU rProteins, light yellow for 16S, purple for capreomycin (CPM) and red for the anticodon stem loop of the P-site tRNA. The cartoon in the inset box is an overlay of the 50S and 70S viewed from the SSU. The handle swings 40° counter-clockwise upon the association between SSU and LSU.

Figure 2.

Interactions of the handle with other ribosomal components within the Mtb ribosomes. (A) Ribbon models showing where the handle (purple) interacts with rProtein uL2 (blue) and H68 (black) when in the 50S alone. The cryo-EM density of the 50S is low-pass filtered to 8Å resolution and overlaid onto the model. Black and blue stars indicate the sites of interactions from the handle to H68 and uL2, respectively. The two eye cartoons, top and bottom, label the cutting plane and viewing direction for Panels C and D, respectively. (B) Ribbon models show the handle interacts with bS6 (orange red) in the 70S. The cryo-EM density for the predominant conformation of the 70S is low-pass filtered to 8Å resolution and overlaid onto the model. Densities of rProteins bL9 and uL2 are colored blue. Cyan and orange stars indicate the sites of interactions from the handle to bL9 and bS6, respectively. The eye cartoon labels the cutting plane and viewing direction of Panel E. (C–E) Cryo-EM densities showing the handle interact with H68, uL2, bS6 and bL9. The 50S is labeled grey with uL2 and bL9 in blue while the handle is in purple. The 30S is in yellow with the rProtein bS6 in orange red. (F) Secondary structure of the handle with arrow heads indicating the nucleotides that interact with H68 (black), uL2 (blue), bS6 (orange) and bL9 (cyan). The sequence, which can form the sarcin-ricin motif, is colored in the red background.

Figure 3.

Correlated motions between the handle, SSU and the L1 stalk in the Mtb 70S ribosome. (A) Mtb 70S in the rotated (left) and non-rotated (right) states. (B) Conformational difference between the rotated and non-rotated states of the Mtb 70S viewing from the solvent exposed side of the SSU. The SSU, L1 stalk, handle and bS6 on the SSU are colored yellow, green, purple and orange red, respectively. Colored arrows indicate the directions and amplitudes of the conformational differences for the handle (purple), the L1 stalk (green), the SSU (yellow) and the bS6 (orange red) on the SSU. (C) Zoom-in view around the handle as viewed from the direction indicated by the eye cartoon in Panel B.

Figure 4.

Unidentified proteins in the Mtb ribosomes discovered near the binding sites for two Mtb translation inhibitors. (A) Cartoon indicating the locations of the mL41-like protein (red), the antibiotic capreomycin (CPM, purple) in the structure of Mtb 70S incubated with capreomycin. Helix h44 of the 16S is in dark yellow. The dashed circle indicates the decoding center. (B) Zoom-in view to show the models of CPM (purple) and polyalanine model of the mL41-like protein (red) and their neighboring environment. SSU protein uS12 (orange), helices h44, h45 of the 16S (yellow) and helices H67, H69 of the 23S (grey) are labeled. (C) Cartoon indicating the locations of the unknown protein (red), the linezolid analog 114 (LZD-114, purple) in the structure of Mtb 50S incubated with LZD-114. The 5S is in green. The H89 is in dark gray. The dashed circle indicates the PTC. (D) Zoom-in view to show the models of LZD-114 (purple) and polyalanine model of the unknown protein (red) and their neighboring environment. H39, H89 of the 23S (gray) and Loop D of the 5S (green) are labeled.