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EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon–anticodon duplex

Nature Structural & Molecular Biology volume 21, pages 817–824 (2014)Cite this article

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Abstract

During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2–mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre–EF-G). Our results suggest that the interactions between the decoding center and the codon–anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2–mRNA movement on the 30S subunit.

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Figure 1: Interaction patterns and conservation of loops I and II.
Figure 2: EF-G mutants containing various tip substitutions inhibit translation.
Figure 3: Loop mutants do not impair factor binding to ribosomes and GTPase activity.
Figure 4: Loop mutants fail to support translocation of tRNA and mRNA.
Figure 5: Loop mutants sample the state of intersubunit rotation but cannot enter the DC.
Figure 6: FRET analyses of 30S head swiveling and working model of EF-G–catalyzed translocation.

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Acknowledgements

We thank members of the P. Zhu laboratory and N. Gao for help and discussion. Y.Q. is supported by the Institute of Biophysics 135 Goal-oriented project, National Laboratory of Biomacromolecules (Institute of Biophysics, Chinese Academy of Sciences), and the State Key Laboratory of Molecular Biology (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). This work was supported by grants from the Ministry of Science and Technology of China (2012CB911000 and 2013CB531200 to Y.Q.), the National Natural Science Foundation of China (31322015, 31170756 and 31270847 to Y.Q.) and the Chinese Academy of Sciences (project KSZD-EW-Z-003 to Y.Q.).

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Authors and Affiliations

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

    Guangqiao Liu, Guangtao Song, Danyang Zhang, Dejiu Zhang, Zhikai Li, Jianshu Dong, Weimin Gong & Yan Qin

  2. Department of Chemical Biology, Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing, China

    Zhixin Lyu & Xin Sheng Zhao

  3. NOXXON Pharma AG, Berlin, Germany

    John Achenbach

  4. Charité, Institute for Medical Physics and Biophysics, Berlin, Germany

    Knud H Nierhaus

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Contributions

G.L., G.S., Danyang Zhang, Dejiu Zhang and Z. Li cloned constructs and performed biochemical assays. Z. Lyu and G.S. collected FRET data and with X.S.Z. analyzed the FRET data. J.D. collected X-ray data and with W.G. resolved the structures. J.A. prepared some figures and analyzed data. Y.Q. and K.H.N. analyzed all data and wrote the manuscript. All authors discussed the results and commented on the manuscript. Y.Q. directed and supervised the project.

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Correspondence to Yan Qin.

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Integrated supplementary information

Supplementary Figure 1 Sequence and structure alignment of EF-G homologs.

(a) Sequence alignments of loops I and II of EF-G and homologues. EF-G, bacterial EF-G; mtEF-G1, mitochondrial EF-G orthologue (translocase); mtEF-G2, mitochondrial paralogue (terminase); Tet(O), members of the Tet(O) family mediating tetracycline resistance. Red, 100% identity of the amino acid sequence with the bacterial consensus sequence of EF-G; blue, conservative amino acid substitution.(b) The overall structure alignment of Tet (O)–GTP conformer on the ribosome (orange, PDB 3J36) in comparison with EF-G–GTP in EF-G–POST (cyan, PDB 2WRI). Domains I-III and V are indicated as common domains. The dashed line surrounded region is the specific domain, i.e. domain IV (D IV). D IVs from EF-G (dark blue) and Tet(O) (dark yellow) are aligned and zoomed-in with 90° turn.

Supplementary Figure 2 In vivo experiments of EF-G and the mutants.

(a) Colonies of E. coli cells over-expressing EF-G or its mutants on agar plates. (b) Polysome pattern of the cells in a. Without IPTG treatment (IPTG-), all mutants exhibited similar polysomes pattern as in WT EF-G cell (black curves). When the cells have been induced with ITPG, the polysomes patterns showing difference are indicated in red (strong) and pink (mild).

Supplementary Figure 3 Binding assays of EF-G and its mutants.

(a) Binding of EF-G or its mutants to vacant ribosomes was analyzed by sucrose cushion ultracentrifugation and SDS-PAGE. Bands corresponding to EF-G and the ribosomal protein S1 are indicated by the arrows. Pre: reaction mix before sucrose cushion. Su/Pe: Supernatant/Pellet after sucrose cushion. (b) The quantification of chemical probing results. TO I is shown as an example (red rectangle). Error bars, s.e.m. (n = 3 technical replicates). **P<0.01, ***P<0.001 by two-tailed Student's t test.

Supplementary Figure 4 FRET analyses of 30S head swiveling.

30S head swiveling indicated by changes in FRET efficiencies between donor Alexa 555 and acceptor Alexa 647 probes attached to S11 and S13 for the S11-Donor/S13-Acceptor pair. PRE 70S complexes were rapidly mixed with EF-G wt or the EF-G mutant S588P or Δloop II and GTP at time 0 and Alexa 647 emission was recorded. Each trace represents the average of 10 reactions.

Supplementary Figure 5 Structural comparison EF-G and D IV loops in solution and on the ribosome.

(a) The overall structure alignment of EF-G in the GTP (PDB 2OM7) and GDP (PDB 2WRI) conformations on the ribosome. When D IVs are aligned and zoomed-in with 90° turn, the tip of loop I moved 10 Å towards P-site upon GTP hydrolysis, whereas loop II remained unchanged. (b) The same proteins of a but in solution. GTP conformer (PDB 1WDT), GDP conformer (PDB 1FNM). The upper tip of D V moved 25 Å. When D IVs were aligned and zoomed-in, the tip of loop I moved 8 Å in the opposite direction compared to its conformation on the ribosome. (c) Structural comparison of EF-G on POST complexes. ecEF-G and ttEF-G are extracted from E. coli and T. thermuphilus POST–EF-G complex (PDB 4KIY and 4KBV), respectively. The overall structures of the two EF-Gs were aligned or D IVs were aligned and zoomed-in with 90° turn. (d) The interface of EF-G with the DC in EF-G–POST (PDB 2WRI). Common domains of EF-G contact only the ribosome whereas D IV interacts directly with tRNA2–mRNA through its tip region. (e) The zoom-in view of the interface between D IV tip with P-tRNA–mRNA or with DC. A-tRNA (yellow) has been extracted from EF-Tu–PRE (PDB 2Y0U).

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Liu, G., Song, G., Zhang, D. et al. EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon–anticodon duplex. Nat Struct Mol Biol 21, 817–824 (2014). https://doi.org/10.1038/nsmb.2869

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