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. 2017 Sep 28;8(1):722.
doi: 10.1038/s41467-017-00718-x.

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy

Linda E Franken  1 , Gert T Oostergetel  1 , Tjaard Pijning  1 , Pranav Puri  2 , Valentina Arkhipova  2 , Egbert J Boekema  1 , Bert Poolman  2 , Albert Guskov  3
Affiliations

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy

Linda E Franken et al. Nat Commun. .

Abstract

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine-Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.When bacteria enter the stationary growth phase, protein translation is suppressed via the dimerization of 70S ribosomes into inactive complexes. Here the authors provide a structural basis for how the dual domain hibernation promotion factor promotes ribosome dimerization and hibernation in bacteria.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
The complete 70S model of L. lactis. a Protein models fitted into the EM density and rotated over 90° with RNA in gray and proteins colored differently. Protein names are based on ref. . b rRNA models fitted into the EM density and rotated over 90°
Fig. 2
Fig. 2
Location of the N- and C-terminal domain of HPFlong. The extra, unmodeled densities in the map, indicated by the boxes of a, correspond to HPFlong N-terminal domain (b and Supplementary Movie 1) and C-terminal domain (c, d and in Supplementary Movie 2; colored gold and blue). d has been rotated 90° with respect to a, c, showing the dimeric interaction of the C-terminal domains. e The surrounding proteins (steel blue) and RNA (orange) of the N-terminal domain. f The linking of the distant N- and C-terminal domains (NtD and CtD) is shown schematically. Their respective locations in the dimer are clarified by the surrounding ribosomal proteins (purple lines) and 16S rRNA (red lines)
Fig. 3
Fig. 3
Different conformational states of 100S ribosomes. Rigid-body fits of two 70S models without the C-terminal domains of HPFlong into the low-resolution maps of the two most extreme conformations, closed state (a, b) and open state (c, d). The difference between the two classes depicts a 55° rotation around the interface. The ‘closed’ conformation closely resembles our 5.6 Å map. Colors indicate 16S rRNA (orange), protein uS2 (cyan and purple) and other proteins (blue). The rotation causes H26 to change its interaction from H26 to protein uS2 from the other ribosome, effectively widening the space between the two 70S ribosomes within the dimer (arrows in b, d)
Fig. 4
Fig. 4
Oligomeric state analysis of HPFlong by SEC-MALLS. The Superdex 200 10/300 column (GE Healthcare) was equilibrated with 100 mM Tris-HCl, 150 mM NaCl (pH 8.0), and the protein was injected in the same buffer. The chromatogram (elution volume is indicated on the x axis) shows the readings of refractive index (RI) detector in black (the scale for the RI detector is shown in the left-hand axis). The thick blue line indicates the calculated molecular mass of the eluting protein throughout the chromatogram (scale on the right-hand axis). The calculated molecular weight is 40.5 kDa (molecular weight of a monomer is 21.3 kDa)
Fig. 5
Fig. 5
Phylogenetic tree of HPF homologs. The tree is based on 110 proteins homologous to HPFlong from L. lactis. The average linker length of HPFlong is given by the number of residues in each of the lineages
Fig. 6
Fig. 6
Ribosome dimerization: RMF vs. HPFlong. a–d Comparison of 3D maps of E. coli (blue ) and L. lactis (yellow) 100S ribosomes. The density for the E. coli ribosome was obtained by taking the maximum voxel value of two copies of the EM-density map from Kato et al.. Two copies of the L. lactis model and two copies of the T. thermophilus structure with RMF (PDB-code 4V8G), thus displaying the mechanism of E. coli, not T. thermophilus, were fitted into the corresponding density (c, d). c Zoom in of dimer interface showing proteins uS2 (marked red for T. thermophilus and cyan for L. lactis). In L. lactis the dimerization process is mediated by the C-terminal domain of HPFlong (green), whereas in T. thermophilus uS2 interacts with proteins uS3, uS4, and uS5 from the opposing ribosome (previously described as second contact site). d The RNA chains of the L. lactis 100S (orange) and T. thermophilus 100S structures (purple). Helix 26 plays a key role in dimerization of L. lactis ribosomes but not in T. thermophilus. e, f Comparison of the 16S rRNA of ribosomes from L. lactis (orange) to T. thermophilus in apo-state (e, blue) and RMF-bound state (f, purple). The 100S map from L. lactis is depicted in gray in the background. By looking at the helices 39 and 40 of the head domain, it is clear that the ribosome from L. lactis is in the apo-state and does not undergo a conformational change upon binding of HPFlong
Fig. 7
Fig. 7
Schematic of dimerization of ribosomes in L. lactis and E. coli. Because HPFlong is a dimer in solution it will collect two ribosomes consecutively (a). Prolonged exposure to an excess of HPFlong shifts the equilibrium toward monomeric inactive ribosomes. In E. coli, (b) each ribosome first collects a copy of HPFshort and RMF. The conformational change upon binding of RMF allows the two ribosomes to dimerize. When YfiA binds at the location of HPFshort, RMF can no longer bind and the ribosomes do not dimerize
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