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EF-Tu
EF-Tu
EF-Tu
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Elongation Factor Thermo Unstable
EF-Tu (blue) complexed with tRNA (red) and GTP (yellow) [1]
Identifiers
SymbolEF-Tu
PfamGTP_EFTU
Pfam clanCL0023
InterProIPR004541
PROSITEPDOC00273
CATH1ETU
SCOP21ETU / SCOPe / SUPFAM
CDDcd00881
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
EF-Tu
Identifiers
SymbolGTP_EFTU_D2
PfamPF03144
InterProIPR004161
CDDcd01342
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Elongation factor Tu domain 3
Identifiers
SymbolGTP_EFTU_D3
PfamPF03143
InterProIPR004160
CDDcd01513
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes.[2][3][4] It is found in eukaryotic mitochondria as TUFM.[5]

As a family of elongation factors, EF-Tu also includes its eukaryotic and archaeal homolog, the alpha subunit of eEF-1 (EF-1A).

Background

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See also: Translation (biology)

Elongation factors are part of the mechanism that synthesizes new proteins through translation in the ribosome. Transfer RNAs (tRNAs) carry the individual amino acids that become integrated into a protein sequence, and have an anticodon for the specific amino acid that they are charged with. Messenger RNA (mRNA) carries the genetic information that encodes the primary structure of a protein, and contains codons that code for each amino acid. The ribosome creates the protein chain by following the mRNA code and integrating the amino acid of an aminoacyl-tRNA (also known as a charged tRNA) to the growing polypeptide chain.[6][7]

There are three sites on the ribosome for tRNA binding. These are the aminoacyl/acceptor site (abbreviated A), the peptidyl site (abbreviated P), and the exit site (abbreviated E). The P-site holds the tRNA connected to the polypeptide chain being synthesized, and the A-site is the binding site for a charged tRNA with an anticodon complementary to the mRNA codon associated with the site. After binding of a charged tRNA to the A-site, a peptide bond is formed between the growing polypeptide chain on the P-site tRNA and the amino acid of the A-site tRNA, and the entire polypeptide is transferred from the P-site tRNA to the A-site tRNA. Then, in a process catalyzed by the prokaryotic elongation factor EF-G (historically known as translocase), the coordinated translocation of the tRNAs and mRNA occurs, with the P-site tRNA moving to the E-site, where it dissociates from the ribosome, and the A-site tRNA moves to take its place in the P-site.[6][7]

Biological functions

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The cyclical role of EF-Tu in translation. Structures are from PDBs 1EFT, 1TUI, and 1TTT.

Protein synthesis

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EF-Tu participates in the polypeptide elongation process of protein synthesis. In prokaryotes, the primary function of EF-Tu is to transport the correct aa-tRNA to the A-site of the ribosome. As a G-protein, it uses GTP to facilitate its function. Outside of the ribosome, EF-Tu complexed with GTP (EF-Tu • GTP) complexes with aa-tRNA to form a stable EF-Tu • GTP • aa-tRNA ternary complex.[8] EF-Tu • GTP binds all correctly-charged aa-tRNAs with approximately identical affinity, except those charged with initiation residues and selenocysteine.[9][10] This can be accomplished because although different amino acid residues have varying side-chain properties, the tRNAs associated with those residues have varying structures to compensate for differences in side-chain binding affinities.[11][12]

The binding of an aa-tRNA to EF-Tu • GTP allows for the ternary complex to be translocated to the A-site of an active ribosome, in which the anticodon of the tRNA binds to the codon of the mRNA. If the correct anticodon binds to the mRNA codon, the ribosome changes configuration and alters the geometry of the GTPase domain of EF-Tu, resulting in the hydrolysis of the GTP associated with the EF-Tu to GDP and Pi. As such, the ribosome functions as a GTPase-activating protein (GAP) for EF-Tu. Upon GTP hydrolysis, the conformation of EF-Tu changes drastically and dissociates from the aa-tRNA and ribosome complex.[4][13] The aa-tRNA then fully enters the A-site, where its amino acid is brought near the P-site's polypeptide and the ribosome catalyzes the covalent transfer of the polypeptide onto the amino acid.[10]

In the cytoplasm, the deactivated EF-Tu • GDP is acted on by the prokaryotic elongation factor EF-Ts, which causes EF-Tu to release its bound GDP. Upon dissociation of EF-Ts, EF-Tu is able to complex with a GTP due to the 5– to 10–fold higher concentration of GTP than GDP in the cytoplasm, resulting in reactivated EF-Tu • GTP, which can then associate with another aa-tRNA.[8][13]

Maintaining translational accuracy

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EF-Tu contributes to translational accuracy in three ways. In translation, a fundamental problem is that near-cognate anticodons have similar binding affinity to a codon as cognate anticodons, such that anticodon-codon binding in the ribosome alone is not sufficient to maintain high translational fidelity. This is addressed by the ribosome not activating the GTPase activity of EF-Tu if the tRNA in the ribosome's A-site does not match the mRNA codon, thus preferentially increasing the likelihood for the incorrect tRNA to leave the ribosome.[14] Additionally, regardless of tRNA matching, EF-Tu also induces a delay after freeing itself from the aa-tRNA, before the aa-tRNA fully enters the A-site (a process called accommodation). This delay period is a second opportunity for incorrectly charged aa-tRNAs to move out of the A-site before the incorrect amino acid is irreversibly added to the polypeptide chain.[15][16] A third mechanism is the less well understood function of EF-Tu to crudely check aa-tRNA associations and reject complexes where the amino acid is not bound to the correct tRNA coding for it.[11]

Other functions

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EF-Tu has been found in large quantities in the cytoskeletons of bacteria, co-localizing underneath the cell membrane with MreB, a cytoskeletal element that maintains cell shape.[17][18] Defects in EF-Tu have been shown to result in defects in bacterial morphology.[19] Additionally, EF-Tu has displayed some chaperone-like characteristics, with some experimental evidence suggesting that it promotes the refolding of a number of denatured proteins in vitro.[20][21] EF-Tu has been found to moonlight on the cell surface of the pathogenic bacteria Staphylococcus aureus, Mycoplasma pneumoniae, and Mycoplasma hyopneumoniae, where EF-Tu is processed and can bind to a range of host molecules.[22] In Bacillus cereus, EF-Tu also moonlights on the surface, where it acts as an environmental sensor and binds to substance P.[23]

Structure

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EF-Tu bound to GDP (yellow) and GDPNP (red), a GTP-like molecule. The GTPase domain (domain I) of EF-Tu is depicted in dark blue, while the oligonucleotide-binding domains II and III are depicted in light blue. Structures are from PDBs 1EFT and 1TUI, for GDP- and GDPNP-bound EF-Tu, respectively.

EF-Tu is a monomeric protein with molecular weight around 43 kDa in Escherichia coli.[24][25][26] The protein consists of three structural domains: a GTP-binding domain and two oligonucleotide-binding domains, often referred to as domain 2 and domain 3. The N-terminal domain I of EF-Tu is the GTP-binding domain. It consists of a six beta-strand core flanked by six alpha-helices.[8] Domains II and III of EF-Tu, the oligonucleotide-binding domains, both adopt beta-barrel structures.[27][28]

The GTP-binding domain I undergoes a dramatic conformational change upon GTP hydrolysis to GDP, allowing EF-Tu to dissociate from aa-tRNA and leave the ribosome.[29] Reactivation of EF-Tu is achieved by GTP binding in the cytoplasm, which leads to a significant conformational change that reactivates the tRNA-binding site of EF-Tu. In particular, GTP binding to EF-Tu results in a ~90° rotation of domain I relative to domains II and III, exposing the residues of the tRNA-binding active site.[30]

Domain 2 adopts a beta-barrel structure, and is involved in binding to charged tRNA.[31] This domain is structurally related to the C-terminal domain of EF2, to which it displays weak sequence similarity. This domain is also found in other proteins such as translation initiation factor IF-2 and tetracycline-resistance proteins. Domain 3 represents the C-terminal domain, which adopts a beta-barrel structure, and is involved in binding to both charged tRNA and to EF1B (or EF-Ts).[32]

Evolution

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Further information: GTPase § Translation factor family

The GTP-binding domain is conserved in both EF-1alpha/EF-Tu and also in EF-2/EF-G and thus seems typical for GTP-dependent proteins which bind non-initiator tRNAs to the ribosome. The GTP-binding translation factor family also includes the eukaryotic peptide chain release factor GTP-binding subunits[33] and prokaryotic peptide chain release factor 3 (RF-3);[34] the prokaryotic GTP-binding protein lepA and its homologue in yeast (GUF1) and Caenorhabditis elegans (ZK1236.1); yeast HBS1;[35] rat Eef1a1 (formerly "statin S1");[36] and the prokaryotic selenocysteine-specific elongation factor selB.[37]

Disease relevance

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Along with the ribosome, EF-Tu is one of the most important targets for antibiotic-mediated inhibition of translation.[8] Antibiotics targeting EF-Tu can be categorized into one of two groups, depending on the mechanism of action, and one of four structural families. The first group includes the antibiotics pulvomycin and GE2270A, and inhibits the formation of the ternary complex.[38] The second group includes the antibiotics kirromycin and enacyloxin, and prevents the release of EF-Tu from the ribosome after GTP hydrolysis.[39][40][41]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

EF-Tu

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from Grokipedia
EF-Tu (Elongation Factor Tu), also known as elongation factor thermo unstable, is a highly abundant GTP-binding protein (GTPase) in bacteria that serves as the primary carrier for aminoacyl-tRNA during the elongation phase of protein synthesis. It forms a ternary complex with GTP and aminoacyl-tRNA, delivering the latter to the ribosome's A-site for codon-anticodon matching, after which GTP hydrolysis triggers the release of EF-Tu and incorporation of the amino acid into the growing polypeptide chain. As the most abundant protein in bacterial cells like Escherichia coli, EF-Tu is essential for efficient translation and constitutes up to 5-10% of total cellular protein. EF-Tu is the prokaryotic elongation factor; its eukaryotic counterpart is eEF1A. Structurally, EF-Tu comprises three flexibly linked domains: domain I (residues 1–200 in E. coli), which contains the GTP-binding site and activity; domain II (residues ~210–300); and domain III (residues ~300–393), forming a compact, flattened triangular shape in the GTP-bound state that opens upon GDP binding. This conformational switch is regulated by the cycle: in the active GTP-bound form, EF-Tu binds tightly; upon ribosomal delivery, GTP hydrolysis—facilitated by ribosomal elements and a catalytic (His84 in E. coli) in switch II—converts it to the inactive GDP-bound form, which has low affinity for tRNA. Recycling occurs via interaction with Ts (EF-Ts), a that promotes GDP release and GTP rebinding, with their complex exhibiting a (Kd) of approximately 1–10 nM in E. coli. Beyond its core translational role, EF-Tu exhibits moonlighting functions, particularly in , where surface-exposed forms act as adhesins by binding host proteins such as and plasminogen, facilitating microbial attachment and immune evasion. It also interacts with bacterial cytoskeletal elements like MreB to influence cell shape and has been implicated in formation and as a (PAMP) recognized by plant immune systems. Due to its indispensability, EF-Tu is a target for antibiotics, including kirromycin and pulvomycin, which stabilize specific conformational states to inhibit . In some species like , EF-Tu exists as a single copy and shows potential as a drug target, with inhibitors like demonstrating binding affinity (Kd = 207 µM).

Discovery and Background

Historical Discovery

The discovery of EF-Tu began in the mid-1960s with studies on soluble factors essential for polypeptide chain elongation in cell-free extracts from . Researchers led by Herbert Weissbach identified a soluble protein fraction that promoted the incorporation of into polypeptides during assays, distinguishing it from and termination factors. These early experiments utilized ribosomal preparations supplemented with , GTP, and synthetic mRNA templates like poly(U), revealing the factor's role in enhancing binding to ribosomes. In 1968, Joan Lucas-Lenard and Fritz Lipmann purified and characterized this GTP-dependent factor, later designated EF-Tu, demonstrating its specific ability to form a stable complex with and GTP, which facilitated delivery to the ribosomal A-site in poly(U)-directed assays. Concurrent work by Weissbach's group confirmed EF-Tu's interaction with GTP, showing stoichiometric binding and stimulation of aa-tRNA utilization in elongation without direct peptidyl transferase activity. These purification efforts involved fractionation and , yielding a protein with a molecular weight of approximately 45 kDa that was heat-labile and essential for GTP hydrolysis-linked steps. Key experiments in the late 1960s and early 1970s employed systems to delineate EF-Tu's function in elongation. For instance, assays with washed ribosomes and fractionated E. coli extracts demonstrated that EF-Tu, in conjunction with EF-Ts, enabled multiple rounds of aa-tRNA binding and translocation, as measured by increased polyphenylalanine synthesis rates upon factor addition. EF-Tu was also isolated from ribosomal washes, indicating a dynamic association with ribosomes during active , where it could be released by high salt buffers without loss of activity upon renaturation. By the 1970s, biochemical studies focused on EF-Tu's GTP binding and hydrolysis mechanisms. David L. Miller and Weissbach reported a ribosome- and aa-tRNA-dependent GTPase activity intrinsic to EF-Tu, with hydrolysis rates accelerating by over six orders of magnitude in the presence of cognate codon-programmed ribosomes, establishing its role as a regulatory switch in elongation fidelity. These findings, derived from nitrocellulose filter-binding assays and thin-layer chromatography for nucleotide products, highlighted EF-Tu's allosteric transitions between GTP-bound (active) and GDP-bound (inactive) states, paving the way for deeper mechanistic insights.

Nomenclature and Occurrence

EF-Tu, or Tu, received its name from early biochemical characterizations that identified it as a "thermo-unstable" component essential for protein synthesis elongation, due to its sensitivity to heat during purification in the late . In , this protein is universally encoded by the tuf gene, a highly conserved locus that has been instrumental in bacterial owing to its sequence stability across species. While EF-Tu is specific to , its functional orthologs in other domains bear distinct names: eEF1A (eukaryotic 1A) in eukaryotes and aEF-1α in , both of which facilitate delivery during but lack the tuf designation. These orthologs share structural and mechanistic similarities with EF-Tu, underscoring a common evolutionary origin despite domain-specific adaptations. EF-Tu is a hallmark of bacterial cells, where it predominates as one of the most abundant proteins, often comprising 5-10% of the total proteome—particularly under conditions of rapid growth that demand high rates of protein synthesis. In many bacteria, especially gamma-proteobacteria like Escherichia coli, the protein is encoded by duplicated genes, tufA and tufB, which arose through gene duplication and provide functional redundancy to support elevated expression levels without compromising fidelity. This duplication is maintained by mechanisms such as gene conversion, ensuring sequence uniformity and robustness in translation machinery. Bacterial EF-Tu variants also exhibit species-specific post-translational modifications that fine-tune its activity and stability; for instance, N-terminal occurs in , shielding the protein from degradation and potentially modulating interactions. Such modifications, alongside the genetic redundancy in certain lineages, highlight EF-Tu's adaptability to diverse bacterial physiologies while preserving its core role in elongation.

Molecular Structure

Domain Organization

EF-Tu is a monomeric protein with a molecular weight of approximately 43 kDa in , encoded by the tuf genes and consisting of 393 residues. The protein adopts a modular architecture comprising three distinct domains: Domain I (residues 1–200), also known as the G-domain, which is responsible for nucleotide binding and ; and Domains II (residues 211–297) and III (residues 299–393), which form β-barrel structures involved in recognition. Domain I features a canonical Rossmann fold with a central six-stranded β-sheet flanked by six α-helices, characteristic of the superfamily. In contrast, Domains II and III each consist of a six-stranded antiparallel β-barrel, with Domain II exhibiting a Greek key topology that contributes to the overall compactness of the protein. Key functional elements within these domains include the Switch I and Switch II regions in Domain I, which are critical for sensing the state and facilitating activation. Switch I spans residues 40–62 and includes conserved motifs such as the P-loop (residues 18–25) and the effector loop, enabling interactions with the γ-phosphate of GTP. Switch II, encompassing residues 80–100, contains a dynamic α- (helix B, residues 84–93 in the GDP-bound form) that repositions upon GTP binding to stabilize the active conformation. In Domain III, the C-terminal α- (residues 385–393) serves as a docking interface, positioning EF-Tu for interactions with ribosomal components during translation. Under physiological conditions, EF-Tu does not oligomerize and functions as a monomer, but it forms a stable ternary complex with GTP and aminoacyl-tRNA (aa-tRNA), where Domains II and III primarily contact the T-arm and acceptor stem of the tRNA, respectively. The first crystal structure of EF-Tu was resolved in 1985 by Jurnak for the partial GDP-bound Domain I at 2.7 Å resolution, revealing the nucleotide-binding pocket with key interactions involving residues like Lys-19 and Asp-21 in the P-loop. Subsequent high-resolution structures, such as the 1.7 Å intact EF-Tu·GDPNP complex from Thermus thermophilus in 1993, provided atomic details of the full domain organization and the solvent-exposed tRNA-binding surface.

Conformational Dynamics

EF-Tu undergoes significant conformational changes driven by the binding and of GTP, which regulate its affinity for (aa-tRNA) and its role elongation. In the GTP-bound state, EF-Tu adopts a closed, compact conformation where domains I, II, and III are closely apposed, forming a stable platform for high-affinity binding to aa-tRNA with a (Kd) of approximately 1-10 nM. This compact arrangement positions the tRNA-binding sites across domains II and III effectively, enabling the formation of the ternary complex EF-Tu·GTP·aa-tRNA essential for delivery to the . Upon GTP hydrolysis to GDP, EF-Tu transitions to an open conformation characterized by a ~90° rotation of domain I away from domains II and III, resulting in a more extended structure with reduced interdomain contacts. This open form exhibits low affinity for aa-tRNA, with a Kd around 10 μM, preventing stable ternary complex formation and facilitating EF-Tu release from the . The nucleotide-dependent switch is allosterically regulated, as the intrinsic activity of EF-Tu is low at approximately 10^{-4} s^{-1}, but can be dramatically accelerated by ribosomal interaction. To recycle EF-Tu, the GDP-bound form binds Ts (EF-Ts), which acts as a (GEF) by displacing GDP and promoting GTP binding, thereby restoring the closed conformation. EF-Ts binds primarily to domains I and III of EF-Tu·GDP, inducing partial opening at the domain I-II interface to accelerate nucleotide exchange by several orders of magnitude. This exchange cycle ensures rapid reactivation of EF-Tu for subsequent rounds of aa-tRNA delivery. Recent cryo-EM studies since 2015 have revealed intermediate conformational states during ternary complex assembly and GTP , highlighting dynamic flexibility beyond the binary GTP/GDP switch. For instance, structures of the EF-Tu·GTP·aa-tRNA complex on the capture partially rotated domain positions and transient tRNA distortions, providing insights into the energy landscape of conformational transitions. These findings underscore how subtle allosteric shifts in domain interfaces coordinate nucleotide and tRNA accommodation.

Role in Translation Elongation

Aminoacyl-tRNA Delivery

During protein synthesis elongation, Tu (EF-Tu) in its GTP-bound form binds to aminoacylated (aa-tRNA), forming the ternary complex EF-Tu·GTP·aa-tRNA, which delivers the charged tRNA to the ribosomal A site. This binding occurs with high affinity, modulated by the esterified and specific tRNA structural elements such as the T-stem base pairs, ensuring efficient complex assembly across different aa-tRNAs. The GTP-bound conformation of EF-Tu enables this stable interaction, positioning the aa-tRNA for subsequent ribosomal engagement. The ternary complex protects the labile ester bond of aa-tRNA from spontaneous by stabilizing an orthoester acid intermediate structure at the aminoacyl linkage, a mediated by interactions in EF-Tu's effector region. Upon delivery, the complex docks at the ribosomal A site, where the anticodon of the tRNA is positioned to base-pair with the mRNA codon, initiating codon recognition. This docking facilitates initial selection, in which the ribosome assesses the codon-anticodon match before proceeding to GTP . The association of the ternary complex with the proceeds rapidly, with a second-order rate constant of approximately 10^8 M^{-1} s^{-1} under physiological conditions, enabling efficient tRNA recruitment during . , the concentration of ternary complexes—typically limited by EF-Tu and GTP availability—constrains the overall speed of elongation, as higher complex levels correlate with faster protein synthesis rates. Error minimization during initial selection relies on kinetic discrimination between cognate and near-cognate tRNAs, where codon-anticodon pairing in the decoding center accelerates GTP on EF-Tu by over 600-fold for matches compared to near-cognate ones (260 s^{-1} vs. 0.4 s^{-1}). This rapid for tRNAs promotes acceptance, while near-cognate complexes dissociate more readily due to slower activation, achieving an initial selection fidelity of about 60-fold without .

GTP Hydrolysis and Ribosome Interaction

The GTPase activity of EF-Tu is dramatically accelerated upon binding of the ternary complex (EF-Tu·GTP·aa-tRNA) to the ribosome, where the sarcin-ricin loop (SRL) of the 23S rRNA interacts with the Switch II region of EF-Tu to induce a conformational change that positions a catalytic water molecule for inline attack on the γ-phosphate of GTP. This ribosome-stimulated hydrolysis follows Michaelis-Menten kinetics, with the rate given by kcat=k0⋅[ribosome]Km+[ribosome]k_{\text{cat}} = k_0 \cdot \frac{[\text{ribosome}]}{K_m + [\text{ribosome}]}, where k0≈103 s−1k_0 \approx 10^3 \, \text{s}^{-1} represents the maximal turnover rate under saturating ribosome concentrations and KmK_m is the Michaelis constant for ribosome binding. The interaction aligns key residues, such as His84 in EF-Tu, to facilitate proton shuttling and phosphate release, ensuring rapid GTP cleavage only after codon recognition stabilizes the complex. Following GTP hydrolysis, EF-Tu undergoes a large-scale conformational rearrangement to the GDP-bound state, which reduces its affinity for aa-tRNA and leads to the release of EF-Tu·GDP from the , thereby allowing the accommodated aa-tRNA to fully enter the peptidyl transferase center (PTC) for formation.00005-8) The GDP-bound EF-Tu remains inactive until EF-Ts binds and catalyzes GDP dissociation, exchanging it for GTP to regenerate the active EF-Tu·GTP form for subsequent elongation cycles. This post-hydrolysis dissociation is essential for efficient tRNA placement, as persistent EF-Tu binding would sterically hinder PTC access. Proofreading enhances fidelity through kinetic partitioning during the EF-Tu cycle: cognate aa-tRNAs predominantly proceed to accommodation after GTP , while near-cognate tRNAs undergo hydrolysis at a similar rate but are more frequently rejected post-hydrolysis due to unstable codon-anticodon interactions, preventing erroneous incorporation. This mechanism achieves discrimination factors of up to 100-fold for near-cognates versus cognates, with rejection occurring primarily during the accommodation step after EF-Tu release. The process relies on the energy from GTP to create a temporal for tRNA scrutiny without direct chemical . Structural insights from cryo-EM studies in the have visualized EF-Tu in complex with the in rotated hybrid states, revealing how the SRL-Switch II contact stabilizes the pre- conformation and how domain rearrangements in EF-Tu facilitate tRNA accommodation post-. These models, resolved at near-atomic resolution, show EF-Tu bridging the A-site tRNA and the 's GTPase-associated center, with rotated subunits correlating to intermediates. Such structures confirm the dynamic interplay between EF-Tu conformational changes and ribosomal states during GTP and tRNA selection.

Additional Biological Functions

Chaperone and Stress Response Roles

Beyond its canonical role in translation, bacterial Tu (EF-Tu) exhibits moonlighting chaperone activity, binding to unfolded polypeptides to prevent their aggregation and promote refolding. This function is mediated primarily by the EF-Tu·GDP form, which forms stable complexes with denatured proteins such as reduced and carboxymethylated and unfolded bovine pancreatic trypsin inhibitor, but shows no affinity for their native counterparts. In vitro assays have demonstrated that EF-Tu suppresses the thermal aggregation of at 43°C, achieving complete inhibition at 5 μM—effective at concentrations approximately 20-fold below its typical cytosolic level of ~100 μM—while also facilitating the reactivation of chemically denatured and α-glucosidase with yields of up to 32% and 24%, respectively. The GTP-bound conformation of EF-Tu further supports this chaperone capability by enabling high-affinity interactions with hydrophobic regions of unfolded chains, as evidenced in studies with denatured rhodanese and other model substrates. This binding stabilizes non-native proteins during refolding, mimicking the action of dedicated chaperones like DnaK, and is enhanced in the presence of GTP cycles facilitated by EF-Ts, which can boost renaturation efficiency to nearly 90% for substrates like rhodanese. Such activity underscores EF-Tu's role as a versatile folding assistant, particularly under conditions where protein denaturation is prevalent. Due to its high cellular abundance, EF-Tu is well-positioned to contribute to stability under stress conditions through its chaperone activity, preventing misfolding and aggregation of vulnerable proteins, as demonstrated . A notable example occurs in , where EF-Tu upregulation following by macrophages—conditions marked by from —enhances bacterial survival by stabilizing damaged enzymes and preventing aggregation. This stress-induced adaptation highlights EF-Tu's broader physiological importance in pathogen resilience, distinct from its interactions with inhibitory agents.

Interactions with Antibiotics and Toxins

EF-Tu serves as a key target for several that disrupt bacterial protein synthesis by interfering with its cycle and interactions during elongation. Kirromycin, a produced by collinus, binds to a cleft at the interface between domains I and II of EF-Tu, locking the factor in a GTP-mimetic conformation even after GTP to GDP. This binding prevents the release of EF-Tu·GDP·aa-tRNA from the , immobilizing it and halting subsequent rounds of elongation. The high-affinity interaction has an IC50 of approximately 70 nM for sensitive EF-Tu variants. Other antibiotics target earlier steps in the EF-Tu cycle. Pulvomycin, isolated from sp., binds in the cleft between domains I and II of EF-Tu·GTP, sterically blocking the association of (aa-tRNA) and inhibiting formation of the ternary complex essential for delivery to the ribosomal A-site. Similarly, GE2270A, a thiopeptide from Planobispora rosea, binds between domains I and III of EF-Tu, disrupting ternary complex assembly and preventing EF-Tu from engaging the , thereby trapping the factor in an inactive state off the . These inhibitors exploit distinct binding pockets on EF-Tu to achieve selective disruption of aa-tRNA delivery. Natural toxins also exploit EF-Tu vulnerabilities, often mimicking mechanisms to inhibit prokaryotic . Fusidic , a steroidal derived from the fungus sp., primarily stabilizes the related EF-G on the after GTP , but its action indirectly impacts EF-Tu function by causing defects in translocation that back up the elongation cycle and deplete available EF-Tu for new rounds of aa-tRNA delivery. This stabilization of EF-G on the leads to persistent blockage, preventing the from proceeding to the next elongation cycle and indirectly depleting available EF-Tu for aa-tRNA delivery, exacerbating translocation defects in sensitive . Bacteria evolve resistance to these EF-Tu-targeted agents through in the tuf genes encoding EF-Tu, which reduce antibiotic affinity without severely impairing function. For instance, the A375T substitution in domain III of EF-Tu decreases binding of kirromycin and related elfamycins, conferring resistance by altering the conformational switch interface critical for . Such have been observed in selections and contribute to multidrug resistance profiles in clinical isolates, particularly in Gram-positive pathogens like , where they compound resistance to frontline s and complicate treatment of infections.

Regulation and Evolution

Regulatory Mechanisms

The activity of elongation factor Tu (EF-Tu) is tightly regulated through nucleotide exchange mechanisms to ensure efficient cycling between its inactive GDP-bound and active GTP-bound states. Ts (EF-Ts) serves as the (GEF) for EF-Tu, catalyzing the release of GDP from EF-Tu·GDP to form the EF-Tu·EF-Ts complex, which exhibits a high-affinity (K_d) of approximately 2 × 10^{-9} M in . This interaction accelerates GDP dissociation by approximately 6 × 10^4-fold compared to the intrinsic rate of EF-Tu alone, enabling rapid reformation of the EF-Tu·GTP complex for subsequent binding. The structural basis of this exchange involves EF-Ts contacting specific helices in EF-Tu to destabilize GDP binding, ultimately releasing EF-Ts upon GTP association and restoring EF-Tu to its active conformation. Post-translational modifications provide additional layers of control over EF-Tu function, particularly in response to environmental stresses. In various bacteria, including , phosphorylation occurs at serine and residues, such as Ser-155 and Thr-118, which decelerates conformational dynamics and modulates EF-Tu activity by inhibiting GTP and ternary complex formation. This modification is reversible and links EF-Tu to nutrient availability, as restores full activity during favorable growth conditions. Feedback mechanisms at the transcriptional level couple EF-Tu abundance to overall rates, especially during stress responses. The stringent response alarmone ppGpp downregulates expression of the tufB , which encodes EF-Tu in E. coli, by inhibiting transcription at the tufB promoter in a , with significant repression observed at concentrations as low as 0.5 mM ppGpp. This adjustment ensures EF-Tu levels match reduced ribosomal capacity under , preventing resource wastage. Additionally, ppGpp acts as a cellular metabolite that fine-tunes EF-Tu activity through , binding EF-Tu with affinities comparable to physiological concentrations (around 10-100 μM) and thereby slowing to coordinate slowdown during quiescence. Non-canonical GTP analogs, such as GDPNP, further exemplify this by stabilizing the GTP-bound state and inhibiting , mimicking stress-induced controls on EF-Tu dynamics.

Evolutionary Conservation

EF-Tu, known as eEF1A in eukaryotes and aEF-1α in , exhibits remarkable evolutionary conservation, reflecting its essential role in translation elongation across all domains of . The G-domain (Domain I), responsible for GTP binding and hydrolysis, shares the canonical fold that traces back to the (LUCA), ensuring functional universality in delivery to the . Overall sequence identity between bacterial EF-Tu and eukaryotic eEF1A is approximately 33%, with even higher similarity in the G-domain, underscoring the preservation of core catalytic residues despite billions of years of divergence. Archaeal aEF-1α displays comparable conservation to both, forming a monophyletic group in phylogenetic reconstructions that supports a single origin in LUCA. Bacterial EF-Tu and eukaryotic eEF1A differ in Domain III, particularly in post-translational modifications and interactions with accessory factors. events, such as the ancient split of the bacterial tuf genes (tufA and tufB), occurred early in eubacterial , enhancing redundancy and expression levels in fast-growing cells; this duplication is absent in most and eukaryotes, where single-copy genes predominate. These duplications likely arose post-LUCA, coinciding with the diversification of prokaryotic lineages and the emergence of oxygenic environments. Phylogenetic analyses of EF-Tu sequences, including multiple alignments from diverse taxa, demonstrate co-evolution with ribosomal components, particularly the sarcin-ricin loop (SRL) of 23S rRNA, where conserved residues in EF-Tu's switch regions align with SRL motifs critical for activation. This interplay suggests coupled evolution of the machinery, with EF-Tu adaptations mirroring ribosomal changes across phyla. Recent studies highlight (HGT) of tuf genes in bacterial pathogens, such as enterococci, facilitating rapid adaptation. Such transfers, detected through incongruent phylogenies, have propagated traits in clinical isolates, emphasizing EF-Tu's role in microbial evolvability.

Biomedical and Disease

Pathogenic Implications

EF-Tu plays a significant role in bacterial by facilitating , , and persistence within host tissues. In Salmonella enterica, EF-Tu interacts with the actin-like protein MreB to modulate bacterial cell shape, which regulates type 3 secretion system activity and enhances and during intestinal . Similarly, in Helicobacter pylori, EF-Tu is upregulated during and secreted extracellularly, where it localizes to the surface of host THP-1 monocytes, promoting bacterial to gastric epithelial cells and contributing to chronic persistence in the stomach. EF-Tu is a major cell wall-associated protein in Mycobacterium leprae, the causative agent of leprosy. In Mycobacterium avium subsp. paratuberculosis, a related pathogen, EF-Tu binds host fibronectin to enhance adhesion and invasion of intestinal cells, exacerbating chronic inflammatory conditions like Johne's disease. As a pathogen-associated molecular pattern (PAMP), extracellular EF-Tu from gram-negative and gram-positive bacteria triggers innate immune responses by engaging Toll-like receptor 2 (TLR2) on host cells, leading to NF-κB activation and pro-inflammatory cytokine production such as TNF-α and IL-6. This interaction contributes to excessive inflammation in sepsis, where EF-Tu released in bacterial membrane vesicles amplifies TLR2-mediated responses, promoting systemic inflammatory syndrome and organ damage during bloodstream infections. EF-Tu variants are associated with resistance in nosocomial pathogens, including isolates from hospital-acquired infections. Mutations in the tuf genes encoding EF-Tu, such as those conferring resistance to elfamycin s like kirromycin, alter activity and reduce drug binding, enabling survival in clinical settings and complicating treatment of urinary tract and bloodstream infections.

Therapeutic Targeting

Therapeutic targeting of EF-Tu focuses on exploiting its essential role in bacterial protein synthesis to develop novel s, particularly amid rising . Elfamycins, a class of natural product-derived inhibitors, bind to EF-Tu and prevent its dissociation from the after GTP hydrolysis, thereby stalling elongation. Kirromycin, a prototypical elfamycin, locks EF-Tu in a GTP-mimetic state on the , inhibiting translation in . Derivatives of related thiopeptides, such as GE2270A, have advanced further; for instance, the semisynthetic analog LFF571 progressed to phase 2 clinical trials for difficile infection, demonstrating a clinical cure rate of 90.6% in treated patients with minimal adverse effects. Another GE2270A derivative, NAI003, shows preclinical promise against Propionibacterium acnes with selective activity (MIC ~0.5 μg/mL) due to conferring resistance in non-target . efforts have identified additional leads, such as MGC-10, which disrupts EF-Tu·tRNA interaction and exhibits potent activity against methicillin-resistant Staphylococcus aureus (MIC 6 μg/mL). Structure-based has leveraged recent high-resolution structures to refine EF-Tu inhibitors. Cryo-EM studies from the 2020s, including time-resolved snapshots of EF-Tu·GTP·aa-tRNA delivery to the at ~3 resolution, reveal dynamic conformational changes that guide inhibitor optimization. These structures highlight binding pockets at the EF-Tu domain interfaces, enabling rational modifications to enhance potency and bacterial specificity, as seen in semisynthetic thiopeptide variants that improve while retaining EF-Tu affinity. Key challenges in EF-Tu targeting include bacterial resistance mechanisms and host selectivity issues. in the tuf , encoding EF-Tu, frequently confer resistance to elfamycins by altering the antibiotic-binding site; for example, single-point mutations like A375T in Escherichia coli EF-Tu reduce kirromycin sensitivity by over 100-fold. Additionally, the high sequence homology (~40-50%) between bacterial EF-Tu and the mitochondrial homolog TUFM raises concerns for off-target effects, potentially disrupting mitochondrial protein synthesis and causing , though early elfamycins like kirromycin exhibit low mitochondrial inhibition. Emerging prospects emphasize synergistic strategies and pathogen-specific applications. Combination therapies pairing EF-Tu inhibitors with ribosome-targeting agents, such as , show enhanced efficacy against multidrug-resistant Gram-positives by simultaneously blocking elongation and peptidyl transferase activity. For , post-2020 research highlights EF-Tu as a viable target in , with patents describing novel small-molecule inhibitors that exploit species-specific EF-Tu variants for improved penetration and reduced resistance potential. Ongoing cryo-EM-guided efforts aim to develop next-generation inhibitors with broader spectrum activity while minimizing mitochondrial risks.

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