Recombinant Elongation factor Ts (tsf)

What is the primary function of Elongation Factor Ts in bacterial translation?

Elongation Factor Ts (EF-Ts) serves as the guanine-nucleotide exchange factor in prokaryotes, catalyzing the recharging of Elongation Factor Tu (EF-Tu) following GTP hydrolysis during the translation elongation cycle. EF-Ts specifically facilitates the exchange of GDP for GTP on EF-Tu, enabling EF-Tu to engage in subsequent rounds of aminoacyl-tRNA delivery to the ribosome. This recycling function is essential for efficient protein synthesis, as it maintains the pool of active EF-Tu molecules available for translation . In contrast to prokaryotic systems, eukaryotic cells utilize Elongation Factor 1B (eEF1B) to perform this nucleotide exchange function for eEF1A (the eukaryotic equivalent of EF-Tu) .

How is the tsf gene organized in bacterial genomes?

The tsf gene, which encodes EF-Ts, is typically organized in an operon with the rpsB gene (encoding ribosomal protein S2) in bacterial genomes. This genomic arrangement has been well-characterized in organisms like Streptomyces coelicolor A3(2) and Streptomyces ramocissimus. Transcription analysis reveals that rpsB and tsf give rise to a bicistronic transcript initiated from a single promoter upstream of rpsB. Interestingly, an attenuator has been identified in the rpsB-tsf intergenic region that creates an approximately 2:1 ratio of rpsB versus tsf transcripts, suggesting a regulatory mechanism that maintains relative stoichiometry between these components .

What expression systems are commonly used for recombinant EF-Ts production?

Recombinant EF-Ts is typically produced using Escherichia coli expression systems. The most common approach involves cloning the tsf gene into cold-inducible expression vectors such as pCold-1 (Takara), which allows for efficient expression of N-terminal His₆-tagged EF-Ts fusion proteins. For optimal expression, E. coli BL21(DE3)pLysS strains are frequently employed as host cells. Expression is typically induced with 1 mM IPTG and ZnSO₄ at lower temperatures (approximately 15°C) for extended periods (24 hours), which enhances proper folding and solubility of the recombinant protein . This temperature-controlled induction strategy is particularly important for maintaining the native structure and function of EF-Ts.

How does phosphorylation affect the interaction between EF-Ts and EF-Tu during cellular stress?

The interaction between EF-Ts and phosphorylated EF-Tu (P~EF-Tu) becomes particularly important during recovery from stress. Research has demonstrated that dephosphorylation of P~EF-Tu by phosphatases like PrpC can reverse the inhibitory effect on GTP hydrolysis, suggesting that this mechanism allows for rapid restoration of translation when favorable conditions return . When designing experiments investigating stress responses, researchers should consider the differential binding properties of EF-Ts to phosphorylated versus non-phosphorylated EF-Tu, and how these interactions may be modulated during various physiological states.

What molecular factors influence the stability of recombinant EF-Ts during purification and storage?

The stability of recombinant EF-Ts is influenced by multiple factors that should be carefully controlled during purification and storage. Temperature sensitivity is a critical consideration, as EF-Ts from different bacterial species exhibit varying thermostability profiles. For instance, EF-Ts from mesophilic organisms like Escherichia coli (optimal growth at ~37°C) demonstrates different stability characteristics compared to those from thermophilic bacteria .

Buffer composition significantly impacts EF-Ts stability, with optimal conditions typically including:

• pH range: 7.0-8.0

• Salt concentration: 100-200 mM NaCl or KCl

• Reducing agents: 1-5 mM DTT or β-mercaptoethanol to maintain thiol groups

• Glycerol: Addition of 10-20% glycerol prevents aggregation during freeze-thaw cycles

Storage of purified recombinant EF-Ts at -80°C in small aliquots with cryoprotectants helps maintain functionality over extended periods. Activity assays measuring the guanine nucleotide exchange capacity of EF-Ts should be performed before and after storage to confirm retention of biological function .

How can single-molecule techniques be applied to study EF-Ts conformational dynamics?

Single-molecule techniques offer powerful approaches to investigate the conformational dynamics of EF-Ts during its interaction with EF-Tu. These methodologies provide insights beyond traditional bulk biochemical assays by capturing transient intermediates and conformational states.

Fluorescence Resonance Energy Transfer (FRET) is particularly valuable for studying EF-Ts dynamics. This approach requires:

• Site-specific labeling of recombinant EF-Ts with donor fluorophores (typically at non-conserved cysteine residues)

• Complementary labeling of EF-Tu with acceptor fluorophores

• Observation of FRET efficiency changes during nucleotide exchange reactions

Additionally, optical tweezers can measure force-dependent interactions between EF-Ts and EF-Tu, providing insights into mechanical aspects of their association and dissociation. These techniques require specialized equipment and careful control experiments to account for potential artifacts introduced by fluorescent labels or tethering strategies.

When designing single-molecule experiments, researchers should consider the known binding interfaces between EF-Ts and EF-Tu, as most residues directly involved in this interaction are highly conserved across species .

What purification strategies yield the highest activity for recombinant EF-Ts?

Optimized purification of recombinant EF-Ts typically employs a multi-step approach to achieve high purity and activity. Based on experimental protocols from the literature, the following strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His₆-tagged EF-Ts. Binding buffer typically contains 20-50 mM Tris-HCl (pH 7.5-8.0), 300-500 mM NaCl, 5-10 mM imidazole, and 5 mM β-mercaptoethanol. Elution is performed with a gradient of 50-300 mM imidazole .

• Intermediate Purification: Ion exchange chromatography (typically Q-Sepharose) to separate EF-Ts from contaminants with similar metal-binding properties but different charge characteristics.

• Polishing Step: Size exclusion chromatography (Superdex 75 or 200) to remove aggregates and achieve homogeneous preparation. Running buffer typically contains 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, and 5 mM β-mercaptoethanol .

• Activity Assessment: Guanine nucleotide exchange assays measuring the ability of purified EF-Ts to accelerate GDP dissociation from EF-Tu.

For optimal results, all purification steps should be performed at 4°C, and protease inhibitors should be included in initial lysis buffers. Tag removal may be necessary for certain applications, though many studies report that N-terminal His₆ tags do not significantly impact EF-Ts function.

How can researchers accurately measure the nucleotide exchange activity of recombinant EF-Ts?

Accurate measurement of EF-Ts nucleotide exchange activity requires careful experimental design and appropriate controls. The following methodological approaches provide quantitative assessment of this activity:

• Fluorescent GDP Displacement Assay:

  • Pre-form EF-Tu- mant-GDP complex (where mant-GDP is a fluorescent GDP analog)

  • Monitor fluorescence decrease as EF-Ts catalyzes replacement of mant-GDP with non-fluorescent GDP

  • Calculate exchange rates under various conditions (temperature, pH, salt)

• Radioactive Nucleotide Exchange Assay:

  • Form EF-Tu- [³H]GDP or EF-Tu- [γ-³²P]GTP complex

  • Add EF-Ts and excess unlabeled nucleotide

  • Measure time-dependent release of labeled nucleotide using filter binding

  • Determine kinetic parameters (kon, koff, Kd)

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding thermodynamics between EF-Ts and EF-Tu

  • Provides complete thermodynamic profile (ΔH, ΔS, ΔG, Kd)

  • Requires higher protein concentrations (typically 15-25 μM in the ITC cell)

Control experiments should include testing nucleotide exchange in the absence of EF-Ts to establish baseline rates. Additionally, comparing the activity of recombinant EF-Ts with that of the native protein (when available) helps validate the functional integrity of the recombinant preparation.

What strategies can overcome solubility issues during recombinant EF-Ts expression?

Solubility challenges are common during recombinant EF-Ts expression, particularly at high expression levels. Several strategies can effectively address these issues:

• Temperature Optimization:

  • Lower induction temperature to 15-18°C

  • Extend expression time to 18-24 hours

  • This approach significantly reduces inclusion body formation

• Co-expression with Chaperones:

  • Co-transform with plasmids expressing molecular chaperones like GroEL/GroES

  • Trigger Factor (TF) co-expression may be particularly beneficial, though it doesn't form stable complexes with EF-Ts directly

• Fusion Tags Beyond His₆:

  • MBP (Maltose-Binding Protein) significantly enhances solubility

  • SUMO tag improves folding while allowing tag removal with specific proteases

• Buffer Additives During Lysis:

  • Non-detergent sulfobetaines (NDSB) at 0.5-1.0 M

  • Low concentrations (0.1-0.5%) of mild detergents like Triton X-100

  • Arginine (50-100 mM) to reduce aggregation

• Refolding Protocols (if inclusion bodies form):

  • Solubilize in 6 M guanidine-HCl or 8 M urea

  • Refold by rapid dilution or dialysis against buffer containing 0.5 M arginine

  • Monitor refolding by measuring nucleotide exchange activity

Systematic screening of these approaches, potentially using small-scale expression trials, allows identification of optimal conditions before scaling up to preparative quantities.

How do the binding properties of EF-Ts differ between various bacterial species?

The binding properties of EF-Ts exhibit significant species-specific variations that reflect evolutionary adaptations to different environmental conditions. Comparative studies of EF-Ts from mesophilic organisms (e.g., Escherichia coli) and thermophilic bacteria (e.g., Bacillus stearothermophilus) reveal important differences in their interactions with nucleotides and EF-Tu .

These binding differences are quantified in the following table:

This differential binding is primarily attributed to variations in the G-domain of EF-Tu across species, which directly interacts with EF-Ts. Notably, the G-domain of Bacillus stearothermophilus maintains binding properties similar to the full-length protein, while the isolated G-domain from E. coli shows dramatically reduced affinity .

These species-specific differences must be considered when designing in vitro reconstitution experiments or when selecting EF-Ts for biotechnological applications requiring specific temperature optima or exchange kinetics.

What structural elements of EF-Ts are essential for its nucleotide exchange function?

The nucleotide exchange function of EF-Ts depends on specific structural elements that have been conserved throughout evolution. Based on crystallographic studies and mutational analyses, the following structural features are essential:

• C-terminal Domain: Contains a conserved motif that directly interacts with the G-domain of EF-Tu, facilitating the initial contact in the exchange process.

• Subdomain N: Contains residues that insert into the nucleotide-binding pocket of EF-Tu, disrupting interactions with GDP and promoting its release.

• Coiled-Coil Domain: Provides structural stability and contributes to the proper positioning of catalytic residues.

• Conserved Interface Residues: Specific amino acids at the EF-Tu/EF-Ts interface are highly conserved across species, highlighting their functional importance in nucleotide exchange .

Mutagenesis studies have demonstrated that alterations in these critical regions significantly impair nucleotide exchange activity. Particularly important are the conserved residues that directly insert into the nucleotide-binding pocket of EF-Tu, as these physically displace GDP and prevent rebinding until EF-Ts dissociates and allows GTP to bind.

Interestingly, in Streptomyces species, a single EF-Ts can interact with multiple and highly divergent EF-Tu proteins (showing only about 65% amino acid sequence identity), suggesting remarkable adaptability in the recognition mechanism .

How does the structure of EF-Ts contribute to its ability to function with multiple EF-Tu variants?

In certain bacterial species, particularly Streptomyces, a single EF-Ts protein can interact with multiple highly divergent EF-Tu variants despite their significant sequence differences. This remarkable adaptability is attributed to specific structural features of EF-Ts:

Experimental evidence from Streptomyces species demonstrates that a single tsf gene product can effectively recycle multiple tuf gene products (encoding different EF-Tu variants), enabling efficient translation despite the unusual presence of multiple divergent EF-Tu species . This adaptability illustrates an elegant evolutionary solution that maintains translation efficiency while allowing diversification of EF-Tu functions.

How do the molecular interactions between EF-Ts and Hsp33 affect protein translation during stress?

Recent research has revealed complex molecular interactions between EF-Ts, EF-Tu, and the molecular chaperone Hsp33 during cellular stress conditions. Hsp33, a redox-regulated holding chaperone, exhibits unfoldase and aggregase activity against EF-Tu even in its reduced state. These interactions have significant implications for protein translation regulation during stress .

Gel filtration and light scattering experiments demonstrate that:

• EF-Tu unfolding and subsequent aggregation induced by Hsp33 occur even when EF-Tu is in complex with EF-Ts, despite EF-Ts normally enhancing EF-Tu stability .

• Trigger Factor (TF), another molecular chaperone, markedly amplifies Hsp33-mediated EF-Tu unfolding and aggregation, as evidenced by increased EF-Tu content in oligomeric fractions from gel filtration of EF-Tu:TF:Hsp33 mixtures compared to EF-Tu:Hsp33 alone .

These interactions represent a stress-response mechanism where translation is regulated through the targeted modification of elongation factors. Researchers investigating translation regulation during stress should consider the dual role of EF-Ts: under normal conditions, it promotes translation by recycling EF-Tu, but during stress, its protective effect against Hsp33-mediated unfolding is limited, allowing for rapid translation attenuation .

What techniques are most effective for detecting conformational changes in EF-Ts upon binding to EF-Tu?

Multiple complementary techniques can effectively detect conformational changes in EF-Ts upon binding to EF-Tu, each providing different insights into the structural transitions:

For optimal results, researchers should employ multiple techniques in parallel, as each provides unique information about different aspects of the conformational changes. Integration of these data provides a comprehensive understanding of how EF-Ts structurally adapts upon engaging with EF-Tu .

How can researchers effectively study the kinetics of EF-Ts:EF-Tu complex formation and dissociation?

Understanding the kinetics of EF-Ts:EF-Tu complex formation and dissociation is crucial for elucidating the regulatory mechanisms of protein translation. Several techniques provide complementary information about these kinetic processes:

• Surface Plasmon Resonance (SPR):

  • Real-time monitoring of association and dissociation phases

  • Determination of kon, koff, and Kd values under various conditions

  • One protein (typically His-tagged EF-Ts) is immobilized on a sensor chip while the other (EF-Tu) flows over the surface

• Biolayer Interferometry (BLI):

  • Similar to SPR but uses optical interference patterns

  • Less sensitive to buffer refractive index changes

  • Particularly useful for determining the effect of nucleotides (GDP/GTP) on complex stability

• Stopped-Flow Spectroscopy:

  • Measures rapid kinetics (millisecond timescale)

  • Can detect conformational changes using fluorescent labels or intrinsic fluorescence

  • Ideal for capturing transient intermediates in the exchange reaction

• Isothermal Titration Calorimetry (ITC):

  • Provides thermodynamic parameters (ΔH, ΔS, ΔG) along with binding constants

  • Experiments typically performed at 20°C with protein concentrations around 15 μM

  • Data analyzed using one-set of sites binding models

Experimental design should account for the influence of:

• Buffer conditions (pH, ionic strength)

• Temperature (especially relevant for comparing mesophilic vs. thermophilic proteins)

• Nucleotide concentrations (GDP/GTP)

• Post-translational modifications (particularly phosphorylation)

The integration of data from these complementary approaches provides a comprehensive understanding of the kinetic and thermodynamic basis of EF-Ts:EF-Tu interactions.