Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions

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Abstract

The signal recognition particle (SRP) recognizes signal sequences of nascent polypeptides and targets ribosome–nascent chain complexes to membrane translocation sites. In eukaryotes, translating ribosomes are slowed down by the Alu domain of SRP to allow efficient targeting. In prokaryotes, however, little is known about the structure and function of Alu domain–containing SRPs. Here, we report a complete molecular model of SRP from the Gram-positive bacterium Bacillus subtilis, based on cryo-EM. The SRP comprises two subunits, 6S RNA and SRP54 or Ffh, and it facilitates elongation slowdown similarly to its eukaryotic counterpart. However, protein contacts with the small ribosomal subunit observed for the mammalian Alu domain are substituted in bacteria by RNA-RNA interactions of 6S RNA with the α-sarcin–ricin loop and helices H43 and H44 of 23S rRNA. Our findings provide a structural basis for cotranslational targeting and RNA-driven elongation arrest in prokaryotes.

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Figure 1: Reconstitution of B. subtilis SRP–ribosome complexes.
Figure 2: Elongation-arrest activity of B. subtilis SRP.
Figure 3: Cryo-EM structures of SRP–RNC complexes.
Figure 4: Isolated densities and models of SRPs.
Figure 5: SRP S domain–ribosome interaction at the ribosome exit.
Figure 6: SRP Alu-domain interaction with the ribosome.

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NCBI Reference Sequence

Protein Data Bank

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Acknowledgements

We thank C. Ungewickell and O. Berninghausen for support with cryo-EM (sample preparation and data collection), E. van der Sluis and J. Musial for E. coli SRP purification, T. Becker for manual data collection and B. Beatrix for support in eukaryotic SRP purification and discussion. We thank R. Matadeen and S. DeCarlo for data collection at the Netherlands Centre for Electron Nanoscopy facility. We thank B. Dobberstein (Ruprecht-Karls-Universitat Heidelberg) for providing tissues. R.B. is supported by the Deutsche Forschungsgemeinschaft (DFG) through grants SFB646, GRK1721 and FOR1805; the Graduate School of Quantitative Biosciences Munich (QBM); the Center for Integrated Protein Science Munich; and the European Research Council (Advanced Grant CRYOTRANSLATION). D.N.W. is supported by the DFG through grants FOR1805, WI3285/3-1 and GRK1721. B.B. is supported by a European Molecular Biology Organization Long Term Fellowship (ALTF 50-2011).

Author information

B.B. prepared the translation extract, the translation arrest and the SRP complexes, and performed single-particle cryo-EM analysis, model building and figure preparation. A.K. performed the MST analysis. All authors interpreted the data and assisted with manuscript preparation. B.B., A.K., D.N.W. and R.B. wrote the manuscript.

Correspondence to Roland Beckmann.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Structural alignment of RNA Alu domains, in vitro reconstitution of BsSRP and microscale thermophoresis assays.

(a) Comparison of the B. subtilis RNA Alu domain with the archaea Pyrococcus horikoshii. A structural superposition of the archaea Pyrococcus horikoshii Alu domain model (blue, PDB entry: 4UYK), onto the B. subtilis RNA Alu domain (red, PDB entry: 4WFM) highlights the highly conserved tertiary fold of the Alu RNA core with an RMSD of 1.25 Å. (b) Size exclusion chromatography diagram of the in vitro reconstitution of BsSRP and SDS-PAGE analysis of the fractions. The different components of BsSRP were either loaded separately or mixed together and subjected to size-exclusion chromatography. From the diagram profile and the SDS-PAGE analysis, Ffh and the 6S RNA co-elute, while HU1 remains unbound. (c) Construct used for the preparation of B. subtilis MifM-stalled RNC carrying FtsQ signal sequence. (d) Purified and labeled (AlexaFluor 488 C5-maleimide) RNCs exposing different nascent chains after SDS-PAGE analysis. (e) Typical MST recording using puromycin-treated empty 70S ribosomes with SRP. (f) Bar diagram representing the association rate constants for different E.coli SRP-RNCs.

Supplementary Figure 2 Cell-extract and time-course translation reaction.

(a) SRP depletion in a control and optimized E. coli cell extract. Western blot signal for the Ffh protein (indicated with an arrow) is reduced to background levels upon ultracentrifugation steps (bottom). No significant changes in protein composition were observed based on Coomassie blue stained SDS-PAGE (top). (b) Membrane reduction in the B. subtilis optimized cell extract. Western blot signal for the RNAse Y membrane protein (indicated with an arrow) is reduced to background levels upon ultracentrifugation steps (bottom). (c-e) Time-course translation reaction. The formation of full length product reporters (FL) encoding TatC or L10 were motored by [35S] Met-labeled in vitro translation in absence or presence of BsSPR or 4.5S SRP and analyzed by Tricin-SDS-PAGE (uncropped gels). (f) Plot representing time-course translation experiments. The synthesis full length product reporter TatC in absence or presence of BsSPR or 4.5S SRP was quantified from 3 independent in vitro translation replicates.

Supplementary Figure 3 Cryo-EM reconstruction and resolution of BsSRP–RNC complexes.

(a) Resolution curve, cryo-EM reconstruction and local resolution of BsRNC-SRP complex using the 8 K CCD detector data. (b) Same as in (a) using K2 direct detector data. (c) Same as in (a) using Falcon II direct detector data.

Supplementary Figure 4 Rigid-body docking of X-ray crystal structure.

(a) Overall representation of B. subtilis Alu domain secondary and 3D structure based on the crystal structure8. (b) Local resolution of BsRNC-SRP cryo-EM reconstruction at the A-site binding factor entry. (c) Rigid body docking of the B. subtilis Alu domain in the BsRNC-SRP cryo-EM reconstruction.

Supplementary Figure 5 SRP models.

(a) BsSRP model fitted into the density with corresponding secondary structure diagram. (b) Mammalian SRP model fitted into the density with corresponding secondary structure diagram.

Supplementary Figure 6 SRP S domain–ribosome interaction at the ribosome exit.

(a) Cryo-EM of the mammalian SRP. Small 30S subunit (yellow), large 50S subunit (grey), P-site tRNA (green), SRP (red). (b) Isolated density of C. familiaris SRP54 at the ribosome exit tunnel. In contrast to the B. subtilis Ffh NG domain, the SRP54 NG domain adopts a more rigid conformation. The signal sequence could be fitted unambiguously into the mammalian SRP54 M domain. (c) Magnified SRP54 M-domain and positioning of the SRP54 eukaryotic-specific C-terminal extension. (d) Isolated density of BsSRP-RNC at the ribosome exit tunnel. Models of BsSRP54 M domain and the signal sequence could be fitted into the density filtered at 8 Å. Isolated density of the signal sequence bound to B. subtilis Ffh M domain is represented in green mesh.

Supplementary Figure 7 Focusing on the Alu domain and blocking of the translation factor–binding site.

(a) Ribosome interactions of the mammalian Alu domain. Density with the fitted model of the mammalian SRP model highlights the interaction mode of the Alu domain with the 80S ribosome by bridging the small and the large subunit at the A-site entry. SRP9/SRP14 interact with h5 and h15 of the 18S RNA. The 7S Alu domain interacts only with uL11. (b) Ribosome interactions of the bacterial Alu domain. SRP density with the 6S model in an orientation similar to (a). In contrast to the mammalian SRP, BsSRP contacts only the large ribosomal subunit. These interactions are mainly mediated by RNA:RNA interactions via docking onto the sarcin-ricin loop. (c) Movement of H43-H44 upon binding of BsSRP. Binding of the BsSPR induces a structural change of H43-H44 from an “open” (cyan) to a “close” state (dark blue). (d) EF-Tu-tRNA-GTP ternary complex at the ribosome translation factor binding site (PDB entry: 2WRO). (e) BsSRP Alu domain binds to the translation factor-binding site (red contour) and thereby competes with elongation factor binding.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1422 kb)

Supplementary Data Set 1

Cell-extract preparation, raw western blot images (PDF 8493 kb)

Overview of the Cryo-EM structure of BsSRP–RNC complex

Cryo-EM reconstruction of the BsSRP–RNC. Small 30S subunit (yellow), large 50S subunit (gray), P-site tRNA (green), 6S RNA (red) and the density corresponding to Ffh M-domain (blue). (MOV 13339 kb)

BsSRP Alu domain interaction with the ribosome

Molecular model of B. subtilis 6S Alu domain 'locking in' by generating a continuous stacking between the α-sarcin-ricin loop and helix H43 H44 of 23S rRNA. (MOV 99290 kb)

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Beckert, B., Kedrov, A., Sohmen, D. et al. Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions. Nat Struct Mol Biol 22, 767–773 (2015) doi:10.1038/nsmb.3086

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