Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH

Abstract

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and upregulates the expression of RafH protein, which is crucial for its survival. The RafH, a dual domain HPF, an orthologue of bacterial long HPF (HPF^long), hibernates ribosome in 70S monosome form, whereas in other bacteria, the HPF^long induces 70S ribosome dimerization and hibernates its ribosome in 100S disome form. Here, we report the cryo- EM structure of M. smegmatis, a close homolog of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The N- terminus domain (NTD) of RafH binds to the decoding center, similarly to HPF^long NTD. In contrast, the C- terminus domain (CTD) of RafH, which is larger than the HPF^long CTD, binds to a distinct site at the platform binding center of the ribosomal small subunit. The two domain-connecting linker regions, which remain mostly disordered in earlier reported HPF^long structures, interact mainly with the anti-Shine Dalgarno sequence of the 16S rRNA.

Fig. 1. 70S ribosome RafH complex.

The 10–40% sucrose density gradient fractionation profile and corresponding peaks analysis on agarose gel stained with Ethidium bromide(0.2 μg/ml) are shown for (a) initial ribosome purification, (b) after dissociation and (c) after re-association. The 30S, 50S, and 70S are labeled for ribosomal small subunit, large subunit, and associated ribosome, respectively. The 23S and 16S are labeled for rRNA of 30S and 50S, respectively. We obtained the same results for all (>5) ribosome preparation. d the 70S ribosomes RafH complex formation and sucrose density pelleting, analyzed on 12% SDS-PAGE, with Coomassie blue staining solution, lane 1 - marker, lane 2 - pure RafH protein, lane 3, 4 - input, lane 5 to 8 – SN (supernatant) and P (pellet) fraction after pelleting on a sucrose cushion. The ribo and bS1 are labeled for ribosome and bacterial ribosomal protein bS1, respectively. e In-vitro protein synthesis assay by titrating ribosome and wild type (WT) RafH, W96A RafH mutant, W111A RafH mutant or antibiotic spectinomycin (SPC) at different stoichiometric ratios of 1:1 or 1:2. The RLU (Relative Luminescence Unit) is measured as the production rate of nLuc activity. Data represents as mean ± SEM (standard error mean), where n = 3. f The 2D cryo- EM micrograph collected during the initial grid screening stage in a JEOL 2200 FS microscope with a Gatan K2 Summit camera. The source data for Fig. 1 is provided in the source data file.

Fig. 2. Cryo- EM structure of Mycobacterium smegmatis 70S ribosome RafH complex.

The overall architecture of the 70S ribosome RafH complex is shown in the mRNA entry site (a) and mRNA exit site (b) by a rotation through a diagonal axis. The SSU 16S rRNA (khaki), SSU r-proteins (dark golden), RafH (maroon), tRNA (pink), the LSU 23S rRNA and 5S rRNA (cornflower blue), LSU r- proteins (royal blue), bS1 (dark salmon) and uS2 (orange) are labeled. The single particle reconstruction data processing summary is shown in Supplementary Fig. 3, gold standard FSC and local resolution of final maps are shown in Supplementary Fig. 4, The cryo- EM maps for individual r-proteins, bS1 and uS2 and E-site tRNA and their model is shown in Supplementary Fig. 5, a full RafH model is shown in Supplementary Fig. 7. c The RafH NTD cryo- EM density (left panel) and model in ribbon (right panel), the top panel is rotated by 180° along X-axis, and shown in the bottom panel, the secondary structures are labeled. d The cryo- EM density in mesh and model in stick style corresponds to RafH linker region residues, 111–124 (maroon), and a-SD (anti-Shine Dalgarno sequence) region of 16S rRNA nucleotides, 1518–1522 (khaki), are shown. For more clarity, an animation is provided in Supplementary Movie 1.

Fig. 3. Ribosome and RafH NTD interaction.

The cryo- EM density in surface view for the small subunit with RafH at the center and its magnified regions, where the cryo- EM density in mesh and model in stick and ribbon are shown. For clarity, the ribosomal large subunit is not shown. The RafH 16S rRNA interactions in counterclockwise, α2 R75 with Bridge B2a (bottom left), α1 with h44 (bottom right), α3 W96 with h23 G673 (middle right), α2 with C1382-C1383 (top right), residues from β2, β3, and β4 with h31 U947, G948 (top left) are shown. For more detail, Supplementary Movies 2–6, Supplementary Fig. 6, and Supplementary Table 2.

Fig. 4. RafH CTD structure and its binding site on the ribosome.

a The RafH CTD binding site present in cryo- EM map in surface style for 70S ribosome RafH complex is shown in the same color scheme used for Fig. 2a, b. A thumbnail for the 70S ribosome is shown on the left. b Cryo- EM density corresponding to RafH CTD in mesh, model in ribbon, and stick is shown. The thumbnail is shown on the left. c The structure of HPF^long CTD dimer (PDB ID; 6T7O) with its first monomer (A) (gray) and second monomer (B) (black) are shown.

Fig. 5. Proposed molecular mechanism for RafH action.

a Inhibition of the translation initiation factor binding by RafH. The pre-translation initiation structure SSU (PDB ID; 5LMT) docked into the ribosome RafH SSU complex structure. For clarity, only the RafH in ribbon (red) with 95% transparent surface, bS1 in ribbon (salmon) with 95% transparent surface, initiation complex factors: mRNA (navy blue), a-SD (gold), IF1 (cornflower blue), IF3 (cyan), and P- tRNA (dark olive green) are shown. A thumbnail is shown in the bottom right. b Protection of 16S rRNA from RNase degradation. The RafH (red) and bS1 (salmon) are shown in ribbon with a 95% transparent surface. 16S rRNA helices, h24, h28, h44, h45, aSD, and 3′ of 16S rRNA are shown in a ribbon with a ladder. The E-tRNA anticodon stem loop (hot pink) is shown in a ribbon with a 95% transparent surface. The RNase nucleolytic site predicted in E. coli 16S rRNA by ref. and corresponding nucleotides in M. smegmatis 16S rRNA are shown in black with the scissors symbol. A thumbnail is shown in the bottom right. The 3′ to 5′ exonuclease RNase PH/RNase R is shown in an orange Pie shape. Its description in 2D is shown in Supplementary Fig. 12.

Fig. 6. Comparison of RafH binding in 70S ribosome with HPF^long binding in 100S ribosome.

a RafH, bS1, uS2, and h40 of 16S rRNA and H54a of 23S rRNA are shown with LSU and SSU in the 95% transparent background. b The corresponding position of HPF^long, uS2, and h40 of 16S rRNA in one of the ribosomes of the Staphylococcus aureus 100S structure (PDB ID; 5NGM) is shown with LSU and SSU in the 95% transparent background. c Two 70S ribosome RafH complex structures docked into the corresponding positions in S. aureus 100S dimer structure (PDB ID; 6FXC) and RafH CTD interacting components are shown in 80% transparent background on the left side and magnified view with a white background are shown in the box on the right side. One 70S ribosome is labeled as A, and the other 70S ribosome is labeled as B. d The HPF^long interacting components uS2 and h40 of 16S rRNA in S. aureus 100S ribosome dimer interface (PDB ID; 6FXC) are shown on the right side with 30S and 50S in 80% transparent background, and a magnified view with white background is shown in the box on the left side. Similar to (c), one 70S ribosome is labeled as A, and the other 70S ribosome is labeled as B. A multiple sequence alignment among HPFs is shown in Supplementary Fig. 9.

Fig. 7. Different modes of ribosome hibernation.

A schematic presentation for the different modes of ribosome hibernation. Top, RafH mediated hibernation in 70S form (from this study). Second from top, HPF^long induces ribosome dimerization and formation of 100S disome^–. Third, from the top, HPF^short and RMF^, induce ribosome dimerization and 100S ribosome formation. Bottom, YfiA hibernates ribosome in the 70S form^,.