Recombinant Elongation factor G (fusA)

What is the primary function of Elongation factor G in bacterial protein synthesis?

Elongation factor G (EF-G) serves two essential functions in bacterial protein synthesis. First, it promotes the translocation step during elongation, where it facilitates the movement of mRNA and tRNAs through the ribosome after peptide bond formation. Second, EF-G works together with ribosome recycling factor (RRF) to dissociate ribosomes from mRNA after the termination of translation .

Mechanistically, EF-G functions as a GTPase, using energy from GTP hydrolysis to drive conformational changes that enable these movements. During translocation, EF-G binds to the ribosome in its GTP-bound state, catalyzes GTP hydrolysis, and undergoes a conformational change that drives the movement of the tRNAs and mRNA through the ribosome .

How does the structure of EF-G relate to its function?

The crystal structure of EF-G (such as that from Staphylococcus aureus resolved to 1.9 Å) reveals a multi-domain protein with dramatic conformational flexibility. EF-G contains five domains (I-V) with domains III-V displaying significant movement relative to domains I-II .

Domain I (also called the G domain) binds and hydrolyzes GTP, containing both switch I and switch II regions that undergo conformational changes upon GTP binding and hydrolysis. Domain IV extends from the protein and is critical for interaction with the ribosomal decoding center. This domain mimics the structure of a tRNA anticodon arm, allowing it to facilitate translocation .

The distinct positioning of these domains, particularly the approximately 25 Å displacement of domain IV relative to domain G observed in S. aureus EF-G compared to T. thermophilus EF-G, highlights the conformational changes essential for EF-G function .

What are the key differences between EF-G's roles in elongation versus ribosome recycling?

In elongation, EF-G catalyzes the movement of peptidyl-tRNA from the A site to the P site and deacylated tRNA from the P site to the E site, along with the coordinated movement of mRNA by one codon. In ribosome recycling, EF-G works with ribosome recycling factor (RRF) to split the ribosome into subunits after termination .

The mechanistic distinction lies in how EF-G interacts with different substrates. During elongation, EF-G interacts directly with tRNAs in the ribosome, while in recycling, EF-G transmits its motor action to RRF through specific surface contacts between domains that mimic the anticodon arm . Kinetic studies suggest that fusidic acid inhibition is more effective during ribosome recycling than during elongation, indicating subtle differences in EF-G's engagement with the ribosome during these processes .

Experimental evidence demonstrates that heterologous EF-G and RRF combinations may be non-functional unless complementary mutations are introduced, suggesting specific interaction surfaces are critical for proper function in ribosome recycling .

How does oxidative stress affect EF-G function, and what are the molecular mechanisms involved?

Elongation factor G is particularly susceptible to oxidation and serves as a critical target during oxidative damage to bacterial translation systems . Research has identified a specific molecular mechanism wherein oxidative stress induces the formation of an intramolecular disulfide bond in EF-G, inactivating the protein.

Specifically, cysteine residue 114 (Cys114) plays a pivotal role in this redox regulation. When oxidized, Cys114 forms a disulfide bond with Cys266, rendering EF-G inactive. Site-directed mutagenesis studies have confirmed this mechanism – the C114S mutant of EF-G remains functional even after H₂O₂ treatment, while wild-type EF-G loses activity .

This redox regulation mechanism appears to be reversible, as thioredoxin can reduce the disulfide bond in oxidized EF-G, restoring its activity. This suggests a potential physiological regulation of protein synthesis in response to changing cellular redox environments .

What approaches can be used to study EF-G conformational changes during translocation?

Investigating EF-G conformational dynamics requires multi-faceted approaches:

How do antibiotics target EF-G, and what mechanisms confer resistance?

Two well-characterized antibiotics that target EF-G are fusidic acid and argyrin B, each with distinct mechanisms of action:

Fusidic Acid (FA): This antibiotic locks EF-G on the ribosome in a post-translocational state with GDP, effectively blocking both elongation and ribosome recycling . FA binds at the GDP/GTP binding pocket and interferes with the post-translocation release of EF-G from the ribosome .

Resistance mechanisms to fusidic acid involve mutations in the fusA gene. Analysis of the S. aureus EF-G structure has revealed that these resistance mutations can be classified as affecting: (1) FA binding directly, (2) EF-G-ribosome interactions, (3) EF-G conformation, or (4) EF-G stability .

Argyrin B: This compound inhibits bacterial protein synthesis by binding directly to EF-G at a novel allosteric binding pocket, distinct from the fusidic acid binding site. Unlike FA, argyrin B binds to free EF-G with high affinity (Kd ~173 nM), potentially blocking its interaction with the ribosome .

Resistance to argyrin B in Pseudomonas aeruginosa and Burkholderia multivorans involves point mutations in fusA1 that result in amino acid substitutions including P414S, S417L, S459F, P486S, T671A, and Y683C, which prevent argyrin B binding .

How can recombinant EF-G be effectively expressed and purified for structural and functional studies?

For high-quality recombinant EF-G production:

• Expression system selection: E. coli BL21(DE3) is commonly used for EF-G expression. For proteins from thermophilic bacteria like T. thermophilus, codon optimization may be necessary.

• Vector design:

  • Include an N-terminal or C-terminal affinity tag (His6, GST, or MBP)

  • Consider TEV or PreScission protease cleavage sites for tag removal

  • Optimize the promoter strength (T7 promoter system works well for EF-G)

• Expression conditions:

  • Induce at OD600 0.6-0.8 with 0.5-1 mM IPTG

  • Lower temperature (16-18°C) for overnight expression often improves solubility

  • Include glucose to prevent leaky expression

• Purification protocol:

  • Metal affinity chromatography (for His-tagged proteins)

  • Ion exchange chromatography to remove nucleic acid contamination

  • Size exclusion chromatography for final purification

  • Include DTT (1-5 mM) in all buffers to prevent oxidation of critical cysteine residues, particularly Cys114, which has been shown to form inhibitory disulfide bonds under oxidizing conditions

• Activity verification:

  • GTPase activity assays

  • Ribosome-dependent translocation assays

  • Binding studies with ribosomes or antibiotics like fusidic acid or argyrin B

What experimental approaches can be used to investigate the interaction between EF-G and ribosome recycling factor (RRF)?

To study EF-G-RRF interactions, researchers can employ several complementary approaches:

• Genetic complementation studies: As demonstrated with T. thermophilus RRF and E. coli EF-G, introducing mutations in EF-G's tRNA-mimic domain or RRF's anticodon arm-mimic region and testing function can reveal interaction surfaces .

• Pull-down assays and co-immunoprecipitation: To detect physical interactions between EF-G and RRF in the presence or absence of ribosomes.

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics between EF-G and RRF, similar to the approach used for studying argyrin B binding to EF-G .

• Cryo-EM structural studies: To visualize the complex of EF-G, RRF, and the ribosome at different stages of recycling.

• Site-directed mutagenesis and chimeric protein analysis: Creating chimeric proteins by swapping domains between EF-G or RRF from different species, as demonstrated in studies where E. coli EF-G with its tRNA-mimic domain replaced by the Thermus domain enabled functionality with Thermus RRF .

• Fluorescence-based assays: To monitor real-time interaction dynamics, particularly to track conformational changes during EF-G-driven RRF action.

How can EF-G overexpression be leveraged for enhanced amino acid production in bacterial systems?

Overexpression of EF-G (fusA) can significantly enhance the production of amino acids in bacterial systems, particularly in Corynebacterium glutamicum. Research has demonstrated that:

Implementation strategy:

• Use strong, inducible promoters for controlled expression

• Balance expression levels of fusA and frr for optimal effect

• Consider co-expressing additional pathway-specific genes

• Optimize fermentation conditions (feeding strategy, pH, oxygen transfer) to maximize production

What methodologies are available for studying the redox regulation of EF-G, and how might this be exploited in research?

To investigate EF-G redox regulation, several methodological approaches can be employed:

• Site-directed mutagenesis: Systematic mutation of cysteine residues (particularly Cys114, Cys266, and Cys398) to serine or alanine to determine their roles in redox sensitivity. The C114S mutation has been shown to render EF-G resistant to H₂O₂-mediated inactivation .

• Thiol modification assays: Using reagents like 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to quantify free thiol groups in EF-G under different redox conditions.

• Mass spectrometry analysis: Peptide-mapping analysis of reduced and oxidized forms of EF-G to identify disulfide bond formation, as demonstrated in studies that identified the Cys114-Cys266 disulfide bond .

• In vitro translation assays: Testing the activity of EF-G under varying redox conditions using reconstituted translation systems, which allows for precise control of the experimental environment.

• Thioredoxin/glutaredoxin interaction studies: Investigating how these cellular redox proteins interact with and regulate EF-G, as thioredoxin has been shown to reduce and reactivate oxidized EF-G .

Research applications:

• Development of oxidation-resistant EF-G variants for biotechnological applications

• Design of expression systems with controlled redox environments for optimal EF-G function

• Exploration of EF-G as a sensor for cellular redox status in bacterial systems

What are the key challenges in distinguishing between EF-G functions in elongation versus ribosome recycling experimentally?

Distinguishing between EF-G's roles in elongation versus recycling presents several experimental challenges:

Methodological approaches to overcome these challenges:

• In vitro reconstituted systems: Using purified components to separately measure translocation versus ribosome recycling activities.

• Specialized kinetic assays: Fast kinetics methods have suggested that fusidic acid inhibition is more effective in ribosome recycling than in elongation, providing one approach to distinguish the functions .

• Selective mutations: Exploiting species-specific compatibility between EF-G and RRF to create chimeric proteins that function in one process but not the other, as demonstrated with T. thermophilus RRF and E. coli EF-G .

• Cryo-EM structural studies: Capturing EF-G in distinct conformational states during elongation versus recycling to identify structural differences that could be exploited experimentally.

How might understanding EF-G structure-function relationships inform the design of novel antimicrobials?

EF-G represents a compelling target for antimicrobial development due to its essential role in bacterial protein synthesis. Understanding its structure-function relationships can inform novel drug design:

• Alternative binding sites: The discovery that argyrin B binds EF-G at an allosteric site distinct from the fusidic acid binding site suggests multiple druggable pockets exist on EF-G . Structure-guided approaches can identify additional potential binding sites.

• Species-specific targeting: Structural differences between bacterial EF-G proteins could be exploited to design species-selective antimicrobials, potentially reducing broad-spectrum resistance development.

• Resistance mechanisms: Analysis of fusA mutations conferring resistance to fusidic acid and argyrin B provides insights into resistance mechanisms, allowing for the rational design of modifications to overcome these resistance mechanisms .

• Dual-function inhibition: Compounds that simultaneously block both elongation and ribosome recycling functions of EF-G could provide enhanced antimicrobial efficacy.

• Conformational trapping: Understanding the conformational dynamics of EF-G during its catalytic cycle opens possibilities for designing molecules that lock EF-G in non-productive conformations, similar to but distinct from fusidic acid's mechanism .