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Structure of the ribosome-EF4 complex. A, overall view of the GTP form of the EF4-ribosome complex. The EF4 protein and 50S and 30S subunits are shown as cryo-EM densities in red, orange, and cyan, respectively. The P and E site tRNAs are shown as cryo-EM densities in lemon and lilac, respectively. The tRNAs are barely visible from this angle. Structural landmarks of the 50S subunit are indicated for clarity. B and C, ribosome ratcheting shown as rotation of the 16S rRNA in the 30S subunit relative to the 50S subunit (viewed from the solvent side of the 30S subunit). The 16S rRNAs of the present complexes of major (B) and minor (C) ribosome populations are shown in cyan and green, respectively. For comparison, the 16S rRNA (gray) in the classical unrotated ribosome with an mRNA and tRNA (26) is shown. The structures are aligned to the 23S rRNA. D, comparison of the 23S rRNAs in the current GTP form of the EF4-ribosome structure (orange) and in the crystal structure of GDP form of the EF4-ribosome complex (14) (gray). The ribosomal L1 and L11 stalks are labeled, and the arrows indicate the direction of the conformational change.
Source publication
Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors, along with
elongation factor G (EF-G) and BPI-inducible protein A (BipA). Although EF4 is highly conserved in bacterial, mitochondrial,
and chloroplast genomes, its exact biological function remains controversial. Here, we present the...
Contexts in source publication
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... with strong densities for EF4 and the P site tRNA were used for the final reconstruction with statistical movie processing to correct the beam-induced particle movements using Relion (24). The final reconstruction yielded a 3.8-Å resolution map, as determined with the gold standard Fourier cell correlation criteria in Relion (supplemental Fig. S1). Initial docking of 30S and 50S subunits (PDB code 5AA0) and EF4 (PDB code 4W2E) x-ray crystal structures into the cryo-EM maps was performed in Chimera (25). We also reconstructed the structure of the same ribosome complex with an counterclockwise rotation at 5.7-Å resolution in a similar ...
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... Structure of the Ribosome-EF4-GDPCP ComplexHere we report the structure of T. thermophilus EF4-GDPCP bound to the ribosome with tRNA in the P and E sites at 3.8-Å resolution, which was reconstructed by single-particle cryo-EM ( Fig. 1A and supplemental Fig. S1). A comparison with the structure of an unrotated ribosome harboring mRNA and tRNAs (26) revealed that the present structure is also unrotated (Fig. 1B), representing a ribosome complex in the POST state, despite the presence of a non-cognate tRNA in the E site whose positioning is virtually identical to the ...
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... Structure of the Ribosome-EF4-GDPCP ComplexHere we report the structure of T. thermophilus EF4-GDPCP bound to the ribosome with tRNA in the P and E sites at 3.8-Å resolution, which was reconstructed by single-particle cryo-EM ( Fig. 1A and supplemental Fig. S1). A comparison with the structure of an unrotated ribosome harboring mRNA and tRNAs (26) revealed that the present structure is also unrotated (Fig. 1B), representing a ribosome complex in the POST state, despite the presence of a non-cognate tRNA in the E site whose positioning is virtually identical to the cognate tRNA in the E site ...
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... the structure of T. thermophilus EF4-GDPCP bound to the ribosome with tRNA in the P and E sites at 3.8-Å resolution, which was reconstructed by single-particle cryo-EM ( Fig. 1A and supplemental Fig. S1). A comparison with the structure of an unrotated ribosome harboring mRNA and tRNAs (26) revealed that the present structure is also unrotated (Fig. 1B), representing a ribosome complex in the POST state, despite the presence of a non-cognate tRNA in the E site whose positioning is virtually identical to the cognate tRNA in the E site (27). Upon closer inspection, we noticed a small ribosome population that was refined to 5.7-Å resolution and displayed a counterclockwise 30S body ...
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... the presence of a non-cognate tRNA in the E site whose positioning is virtually identical to the cognate tRNA in the E site (27). Upon closer inspection, we noticed a small ribosome population that was refined to 5.7-Å resolution and displayed a counterclockwise 30S body rotation with respect to the 50S subunit as well as 30S head swiveling (Fig. 1C). The unrotated and slightly counterclockwise rotated states of our structures are different from the notable clockwise rotation (30S body rotation by 5°) that was recently reported in the crystal structure of EF4-GDP (14) and the cryo-EM reconstitution of the minor population of EF4-GDPNP (a non-hydrolysable GTP analog) bound to the ...
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... in the structures of the GDP and GTP forms. The tip of H23 (the helices of the 50S and 30S subunits are labeled with H and h, respectively) in the L11 region and H78 in the L1 stalk shift away from the A site finger (H38 in 23S rRNA) and the E site tRNA by 18 and 6 Å, respectively, compared with the structure of EF4-GDP bound to the ribosome (Fig. ...
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... electron density map clearly shows both the P site tRNA and EF4 (supplemental Fig. S1B). We were able to build a complete model for EF4, including the regions that are crucial for its function: switch 1 (SW1, residues 38 -60), switch 2 (SW2, residues 81-104), and the unique CTD (residues 490 -610). Although the two switch regions, SW1 and SW2, had been visualized at low resolution based on homology modeling using EF-G ...
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... that the relatively stable binding of the GDP form of EF4 to the ribosome could be a result of the engineered fusion strategy (14). EF4 is capable of binding to the ribosome in the presence of both A and P site tRNAs (PRE) or the P site tRNA only (POST) (2, 10, 12). The overall arrangement of the GTP form of EF4 bound to the POST state is virtually identical to that of the PRE state (supplemental Fig. S3A), according to the recently reported cryo-EM study of E. coli EF4 bound to the ribosome (19). ...
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Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. Where and how in the elongation cycle +1-frameshifting occurs remains poorly understood. We captured six ~3.5-Å-resolution cryo-EM structures of ribosomal elongation complexes formed with the GTPase elongation factor G (EF-G). Three st...
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Citations
... Our results provide no supporting evidence for 4-5° clockwise rotation of the small ribosomal subunit relative to the large subunit observed in X-ray crystal and cryo-EM structures of LepAribosome complexes [21,22]. However, our results are consistent with the cryo-EM reconstruction of LepA bound to NR ribosomes [47]. The ability of EF-G to interact with both NR and R conformations is also important for inducing translocation of mRNA and tRNAs on the 30S subunit. ...
... Our results provide no supporting evidence for 4-5 • clockwise rotation of the small ribosomal subunit relative to the large subunit observed in X-ray crystal and cryo-EM structures of LepA-ribosome complexes [21,22]. However, our results are consistent with the cryo-EM reconstruction of LepA bound to NR ribosomes [47]. ...
Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.
... Furthermore, a paper published during our manuscript preparation revealed that bacteria can remodel their protein expression relevant to biofilm formation in a temperature-dependent manner by modulating BipA abundance (Del Peso Santos et al., 2021). With the desire to support the ongoing effort in BipA-related studies and to expand our efforts on structural study of ribosome-associated proteins and biofilm formation as well as its relevant pathogenesis and resistance (Tanaka et al., 2008;Selmer et al., 2012;Kumar et al., 2015;Yu et al., 2015;Ero et al., 2016;Kumar et al., 2016;Yang et al., 2017), here we report additional evidence to demonstrate the role of BipA in ribosome biogenesis, specifically in large subunit 50S maturation, and conditional protein translation at suboptimal temperature through combinative approaches. In particular, our findings by tandem mass tag (TMT)-based quantitative proteomic analysis identified proteins relevant to RNA metabolism (e.g., DeaD) with increased expression levels upon BipA deletion, such an effect can be suppressed by a further mutation of RluC or complementation by BipA. ...
BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (Δ bipA ) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the Δ bipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.
... Red and blue colour indicated negative charge and positive charge respectively whereas white colour indicates neutral charge positive potential area in the A-site of the 70S ribosome complex that was mainly contributed by S12, L11, and S19 proteins. Biochemical and structural studies have shown that elongation factors; EF-Tu [28][29][30], EF-G [31][32][33], EF-4 [34], EF-P [35] interact with L11 protein, which is found to have the positive potential [27]. This positive potential contributed by these proteins of the A-site may be necessary for the interaction as it has been found that mutant lacking L-11 is extremely compromised in E. coli [36]. ...
Background
Protein synthesis is a cellular process that takes place through the successive translation events within the ribosome by the event-specific protein factors, namely, initiation, elongation, release, and recycling factors. In this regard, we asked the question about how similar are those translation factors to each other from a wide variety of bacteria? Hence, we did a thorough in silico study of the translation factors from 495 bacterial sp., and 4262 amino acid sequences by theoretically measuring their pI and MW values that are two determining factors for distinguishing individual proteins in 2D gel electrophoresis in experimental procedures. Then we analyzed the output from various angles.
Results
Our study revealed the fact that it’s not all same, or all random, but there are distinct orders and the pI values of translation factors are translation event specific. We found that the translation initiation factors are mainly basic, whereas, elongation and release factors that interact with the inter-subunit space of the intact 70S ribosome during translation are strictly acidic across bacterial sp. These acidic elongation factors and release factors contain higher frequencies of glutamic acids. However, among all the translation factors, the translation initiation factor 2 (IF2) and ribosome recycling factor (RRF) showed variable pI values that are linked to the order of phylogeny.
Conclusions
From the results of our study, we conclude that among all the bacterial translation factors, elongation and release factors are more conserved in terms of their pI values in comparison to initiation and recycling factors. Acidic properties of these factors are independent of habitat, nature, and phylogeny of the bacterial species. Furthermore, irrespective of the different shapes, sizes, and functions of the elongation and release factors, possession of the strictly acidic pI values of these translation factors all over the domain Bacteria indicates that the acidic nature of these factors is a necessary criterion, perhaps to interact into the partially enclosed rRNA rich inter-subunit space of the translating 70S ribosome.
... Both BipA and LepA bind in a similar manner to the A-site of the 50S ribosomal subunit. BipA is located in proximity to L6 (Fig. S5) (52), and LepA not only makes direct contact with L6, but also incorporates L10 and L12 ribosomal proteins via its nucleotide-independent chaperone activity (46,53). More recently, ΔlepA strain was cold-sensitive and shown to accumulate the precursor 17S rRNA and 30S particles missing several ribosomal proteins, suggesting a novel 30S ribosome assembly GTPase (54). ...
BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA's role in ribosome biogenesis, here we compared and analyzed the ribosomal protein compositions of MG1655 wild-type and bipA-deletion strains at 20°C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20°C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20°C, suggesting that it encodes a cold shock-inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20°C. Importantly, GFP and α-glucosidase refolding assay revealed that BipA has chaperone activity. Our findings support that BipA is a cold shock-inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.
... A recent study proposed that EF4 functions in the biogenesis of the 30S ribosomal subunits because 30S particles accumulate (11). Structures of ribosome-bound EF4 have been visualized with both pretranslocation (PRE) and posttranslocation (POST) ribosomal complexes (14)(15)(16)(17)(18). ...
Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study we show that growth of an Escherichia coli strain is more susceptible to tetracycline than its EF4 knockout strain. In consistence with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Together, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.
... The antibiotic was shown to cross-link in vivo to A-2602 of the 23S rRNA, placing its site of action in the peptidyl transferase center (PTC), as well as to ribosome-bound LepA, indicating that LepA binds to the ribosome and therefore may be involved in some step of translation. Molecular modeling of the specific binding site of oxazolidinone antibiotics placed it within the A site (34), which is in accordance with the experimentally determined binding site of LepA (15,23,25,27,35). ...
Protein synthesis, the translation of mRNA into a polypeptide facilitated by the ribosome, is assisted by a variety of protein factors, some of which are GTPases. In addition to four highly conserved and well-understood GTPases with known function, there are also a number of non-canonical GTPases that are implicated in translation but whose functions are not fully understood. LepA/EF4 is one of these non-canonical GTPases. It is highly conserved and present in bacteria, mitochondria and chloroplasts, but its functional role in the cell remains unknown. LepA's sequence and domain arrangement is very similar to other translational GTPases, but it contains a unique C-terminal domain (CTD) that is likely essential to its specific function in the cell. Three main hypotheses about the function of LepA have been brought forward to date: 1) LepA is a back-translocase; 2) LepA relieves ribosome stalling or facilitates sequestration; and 3) LepA is involved in ribosome biogenesis. This review will examine the structural and biochemical information available on bacterial LepA and discuss it on the background of the available in vivo information from higher organisms in order to broaden the view regarding LepA's functional role in the cell and how the structure of its unique CTD might be involved in facilitating it.
... Several ribosome-bound LepA structures have been reported (25,(42)(43)(44). In all cases, mRNA and tRNAs were also part of the complex, and the data were often interpreted with the assumption that LepA acts during elongation. ...
Significance
The translational GTPase LepA is a highly conserved bacterial protein whose role in the cell has been elusive. Here, we show that the function of LepA lies in biogenesis of the 30S subunit of the ribosome, rather than in translation elongation, as previously supposed. Loss of LepA results in the accumulation of immature 30S particles lacking certain proteins of the 3′ (head) domain and containing precursor 17S rRNA. The GTPase activity of LepA, like that of other translational GTPases, is stimulated by interactions with both subunits of the ribosome. This implies that LepA acts at a late stage of assembly, in the context of the 70S ribosome.
... Single-site mutation or deletion of this region showed severely reduced back-translocation efficiency. The disruption of the peptidyl-tRNA-P-loop interaction by R560 tip was partially observed in the recent Cryo-EM structure of T. thermophiles POST-EF4 complex, 18 but not in the previous crystal structure. 6 However, when comparing the structural parameters of all 3 POST-EF4 complexes (Table 1), we could find that the 2 structures 6,18 are not real POST complex. ...
... The CTD of EF4 exhibits highly extended contacts with the CCA arm of A/4 site tRNA (Fig. 3B). Together with our publication, 7,17,18 , all the EF4-ribosome structures confirm that EF4 stabilizes the PRE complex via its CTD region. The stabilization of PRE complex by EF4 may explain the recent results of smFRET study, which suggests that EF4 preferentially targets ribosome in the PRE instead of POST state. ...
In the translating ribosomal complex, transfer RNA (tRNA) is stabilized in the ribosome by its anticodon stem-loop (ASL) and 3'-CCA end through base-pairing interactions with mRNA codon on the small subunit and ribosomal RNA in the peptidyl transferase centre (PTC) of large subunit, respectively.Elongation factor 4 (EF4), a highly conserved translational GTPase, has been identified to trigger back-translocation. Early this year, we reported high resolution cryo-EM structures of EF4 in complex with Escherichia coli 70S ribosome in pre- and post-translocational states with direct observations that EF4 disrupts the base pairs between the 3'-end of peptidyl-tRNA and the P-loop of rRNA in PTC. Here, we focus on the novel molecular mechanism how EF4 catalyses back-translocation, and discuss the common and specific energy barriers for forward- and back-translocation.
... 23 Consistent with the universal trGTPase-binding mode, EF4 forms extensive contacts with both 30S and 50S subunits. The overall conformation of EF4-GDP bound to the ribosome in the crystal structure 47 is similar to that of EF4-GDPNP 23,27 and EF-4-GDPCP 68 in complex with the ribosome in cryo-EM reconstructions. It should be noted, however, that unlike the EF4-GDP-ribosome crystal structure 47 and the EF4-GDPCPribosome cryo-EM reconstitution 68 with tRNAs in classical P site and empty A site, the cryo-EM reconstitutions of the EF4-GDPNP-ribosome complexes reveal 2 tRNAs. ...
... The overall conformation of EF4-GDP bound to the ribosome in the crystal structure 47 is similar to that of EF4-GDPNP 23,27 and EF-4-GDPCP 68 in complex with the ribosome in cryo-EM reconstructions. It should be noted, however, that unlike the EF4-GDP-ribosome crystal structure 47 and the EF4-GDPCPribosome cryo-EM reconstitution 68 with tRNAs in classical P site and empty A site, the cryo-EM reconstitutions of the EF4-GDPNP-ribosome complexes reveal 2 tRNAs. In addition to the classical P site, tRNA can also be seen to occupy the A site but in a previously unseen distorted conformation (named A/L and A/4 tRNA in 23 and, 27 respectively) with acceptor arm shifted away from PTC. ...
... 27 Interestingly, a cryo-EM analysis of the EF4-GDPCP-ribosome complex revealed, in addition to unrotated ribosomes, a small population of anti-clockwise rotated ribosomes, whereas clockwise-rotated ribosomes were not observed. 68 Therefore, the significance of ribosome rotation for EF4 functioning is still unclear and requires further studies. ...
EF-G, EF4, and BipA are members of the translation factor family of GTPases with a common ribosome binding mode and GTPase activation mechanism. However, topological variations of shared as well as unique domains ensure different roles played by these proteins during translation. Recent X-ray crystallography and cryo-electron microscopy studies have revealed the structural basis for the involvement of EF-G domain IV in securing the movement of tRNAs and mRNA during translocation as well as revealing how the unique C-terminal domains of EF4 and BipA interact with the ribosome and tRNAs contributing to the regulation of translation under certain conditions. EF-G, EF-4, and BipA are intriguing examples of structural variations on a common theme that results in diverse behavior and function. Structural studies of translational GTPase factors have been greatly facilitated by the use of antibiotics, which have revealed their mechanism of action.
In bacteria, the assembly factors tightly orchestrate the maturation of ribosomes whose competency for protein synthesis is validated by translation machinery at various stages of translation cycle. However, what transpires to the quality control measures when the ribosomes are produced with assembly defects remains enigmatic. In Escherichia coli, we show that 30S ribosomes that harbour assembly defects due to the lack of assembly factors such as RbfA and KsgA display suboptimal initiation codon recognition and bypass the critical codon–anticodon proofreading steps during translation initiation. These premature ribosomes on entering the translation cycle compromise the fidelity of decoding that gives rise to errors during initiation and elongation. We show that the assembly defects compromise the binding of initiation factor 3 (IF3), which in turn appears to license the rapid transition of 30S (pre) initiation complex to 70S initiation complex by tempering the validation of codon–anticodon interaction during translation initiation. This suggests that the premature ribosomes harbouring the assembly defects subvert the IF3 mediated proofreading of cognate initiation codon to enter the translation cycle.
