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HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions

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Abstract

Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock–induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

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Figure 1: HflX is a heat-shock protein and is required for cell survival upon heat stress.
Figure 2: HflX binds to the ribosome in vivo and in vitro, with a preference for the 50S subunit.
Figure 3: HflX dissociates vacant 70S ribosomes in the presence of GTP or GMP-PNP.
Figure 4: HflX efficiently promotes breakdown of puromycin-treated polysomes and splits the PTL.
Figure 5: Cryo-EM structure of the 50S–HflX–GMP-PNP complex.
Figure 6: Specific electrostatic interactions between HflX and the 50S subunit.
Figure 7: Orientation of HflX relative to tRNAs and release factor 2 on the 50S subunit.
Figure 8: Proposed model for the action of HflX during the heat-shock response.

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Acknowledgements

This work was funded by grants from the National Natural Science Foundation of China (31170677 and 31422016 to N.G.) and the Ministry of Science and Technology of China (2013CB910404 to N.G.). S.S. acknowledges research funding from the Swedish Research Council (2010-2619 (M), 2011-6088 (NT), 2014-4423 (NT) and 2008-6593 (Linnaeus grant to Uppsala RNA Research Center); and the Knut and Alice Wallenberg Foundation (KAW 2011.0081 to RiboCORE platform)). We thank the National BioResource Project of Japan for providing E. coli strains BW25113 and JW4131. We also thank the China National Center for Protein Sciences (Beijing) and Tsinghua National Laboratory for Information Science and Technology ('Explorer 100' cluster system) for providing computational resources.

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Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Yanqing Zhang, Wei Cao, Ningning Li, Yixiao Zhang, Xiaoxiao Zhang, Jianlin Lei & Ning Gao

  2. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Chandra Sekhar Mandava & Suparna Sanyal

  3. Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Xiaojing Li & Kaixia Mi

  4. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Dejiu Zhang & Yan Qin

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  1. Yanqing Zhang
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Contributions

J.L., S.S. and N.G. designed experiments. Yanqing Zhang performed quantitative PCR, western blotting, cell growth experiments (with X.L. and K.M.), WT and mutant protein preparation (with X.Z.), ribosome purification, SDGC-based experiments, polysome profile analysis (with D.Z. and Y.Q.), cryo-EM data collection (with J.L.) and image processing (with W.C., N.L., Yixiao Zhang and N.G.). C.S.M. performed kinetics experiments. Y.Z., C.S.M., S.S. and N.G. prepared the manuscript; all authors approved the final manuscript.

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Correspondence to Jianlin Lei, Suparna Sanyal or Ning Gao.

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Integrated supplementary information

Supplementary Figure 1 Growth curves of wild-type and hflX-knockout strains.

Growth curves of WT (wild type) and ∆hflX (KO) cells cultured at 30 °C (a) or 45 °C (b). Data shown are means and s.d. (n = 3 cell-culture replicates).

Supplementary Figure 2 An excess of HflX splits 70S ribosomes in vitro and in vivo.

(a-b) The splitting of purified 70S (0.3 μM) (a) or crude 70S ribosome in cell lysate (freshly prepared by ultra-sonication) (b), with 20-fold excess of HflX in the absence (Apo) or presence of different nucleotides (GMPPNP, GTP, and GDP, 1 mM) was checked by SDGC. (c) Overexpression of HflX from the hflX-pBAD plasmid with 1% L-arabinose resulted in slower growth rate of the bacterial host E. coli BW25113 (WT). The vector alone was treated similarly as a control. Data shown are means and s.d. (n= 3 cell-culture replicates). (d) Polysome profile of the mid-log phase E. coli BW25113 cells without (0%) and with induction of HflX by adding 1 % or 2 % (w/v) L-arabinose (L-ara). While HflX overexpression leads to the increase in the 50S fraction. (e) SDS-PAGE and western blotting analysis (anti-his) to check the expression level of recombinant HflX in the above cells. P, empty pBAD vector; H, pBADhflX plasmid. The bands corresponding to HflX are indicated as asterisks.

Supplementary Figure 3 GTP hydrolysis is essential for release of HflX from the 50S subunit.

(a) Formation of 70S ribosome by association of vacant 30S (0.25 µM) with 50S (0.25 µM) in the absence (brown right triangle), or presence of HflX–GTP (black square) and HflX–GMPPNP (olive green down triangle), followed by increase in Rayleigh light scattering with time in a stopped flow apparatus. (b) The effect of HflX–GTP (black square) and HflX–GMPPNP (olive green down triangle) on the formation of 70S initiation complex. The experimental setup and data fitting were same as in a, except that an mRNA programmed 30S pre–IC was used instead of vacant 30S. As a control, the association reaction was also run without HflX (brown right triangle).

Supplementary Figure 4 Resolution of the cryo-EM density map and conformational changes of the 50S subunit upon HflX binding.

(a) Gold-standard Fourier Shell Correlation (FSC) curve. When FSC is 0.143, resolution of cryo-EM density map of the 50S–HflX–GMPPNP complex is 4.5 Å. (b) Local-resolution map of the cryo-EM map. (c) Histogram of local resolution in terms of individual voxels. (d) The atomic model of the 50S–HflX–GMPPNP complex is displayed in cartoon representation (rRNA colored marine and r-proteins colored green as above), superimposed with the model of the 50S subunit from a crystal structure of empty 70S ribosome (PDB id: 2AWB) (rRNA colored red and r-proteins colored yellow)1. (e-j) Close-up views of the local conformational changes on respective rRNA helices as boxed in d. The displacements of the helices upon HflX binding are marked with arrows.

Supplementary Figure 5 Comparison of HflX with translation factors on the 50S subunit.

(a) A thumbnail of the 50S–HflX–GMPPNP structure in the top left corner and superimposition of HflX with canonical translation factors on the 50S subunit. HflX is colored magenta and other translation factors are colored blue. IF2 (PDB id:1ZO1)2, EF-Tu (PDB id: 2XQD)3, EF-G (PDB id: 2WRI)4, LepA (PDB id: 4W2E)5, RF1 (PDB id: 3D5A)6, RF2 (PDB id: 3F1G)7, RF3 (PDB id: 3SFS)8, RRF (PDB id: 4GD1)9, and YaeJ (PDB id: 4DH9)10. (b) Relative orientation of the GTPase domain (yellow) of HflX and known translation factors to the SRL of the 23S rRNA. For clarification, only the GTPase domains of translation factors are shown. PDB codes of the GTPases shown are same as in a.

Supplementary Figure 6 Point and truncation mutations of HflX affect the 70S-spliting activity of HflX.

(a) The splitting of vacant 70S ribosomes, with two truncation mutations of HflX (in the presence of GMPPNP) determined by SDGC, with a comparison to wild type HflX. (b) HflX double point mutations (R49A-K50A, K55A-K62A, R164A-R165A, R168A-R170A, R185A-R189A, R192A-K194A) significantly impairs the 70S-splitting activity. (c) Binding of mutant forms of HflX to the 50S subunits in the presence of GMPPNP examined using co-sedimentation assay. (d) Control experiments for (c) run without 50S subunits. Bands of HflX are indicated by asterisks.

Supplementary Figure 7 Overexpressing the RRF-encoding gene partially restores the growth of ∆hflX cells under heat-stress conditions.

(a-b) Spotting assay of E. coli strains (WT-pBAD, ∆hflX-pBAD, ∆hflX-pBADfrr, ∆hflX-pBADhflX) without (a) or with heat treatment at 50°C for 30 min (b). Spotting assay data in a and b are representative of three individual experiments. (c) Thermo-killing curves of bacteria incubated at 50°C and detected at different time points. Data shown are means and s.d. (n= 3 cell-culture replicates). WT, E. coli BW25113.

Supplementary Figure 8 Distribution of HflX in ribosomal fractions at normal or high-temperature conditions.

Polysome profile analysis of wild type cells cultured at 30°C (a) or 45°C (b). Distribution of HflX in sedimentation fractions was detected using Western blotting.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note (PDF 1563 kb)

Supplementary Data Set 1

Original blots for Figure 1b (PDF 134 kb)

Cryo-EM density map of the 50S-HflX-GMP-PNP complex (4.5 Å)

The movie shows surface presentation of the cryo-EM density map of the 50S-HflX- GMP-PNP complex (4.5 Å), with atomic model superimposed. Also, a close-up view of interactions between HflX and PTC region of the 50S subunit is shown. (MP4 45791 kb)

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Zhang, Y., Mandava, C., Cao, W. et al. HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions. Nat Struct Mol Biol 22, 906–913 (2015). https://doi.org/10.1038/nsmb.3103

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