Recombinant Thermotoga petrophila Ribosome-recycling factor (frr)

What is Thermotoga petrophila and how does it relate to other Thermotoga species?

Thermotoga petrophila is a hyperthermophilic bacterium isolated from oil reservoir production fluid in Niigata, Japan. It has an optimal growth temperature of 80°C and shares 99.2% 16S rRNA identity with the well-studied T. maritima, indicating their close evolutionary relationship . T. petrophila contains approximately 1,785 protein-coding open reading frames (ORFs).

Comparative genomic analysis places T. petrophila among several closely related Thermotoga species:

Like other Thermotoga species, T. petrophila can utilize various polysaccharides for growth, including xylan, starch, and glucomannan, though it lacks the ability to grow on crystalline cellulose due to the absence of multifunctional enzymes with both endoglucanase and carbohydrate binding module domains .

What is the ribosome-recycling factor (RRF) and what is its functional role in protein synthesis?

The ribosome-recycling factor (RRF) is an essential protein in prokaryotes that, together with elongation factor G (EF-G), catalyzes the recycling of ribosomes after a completed round of protein synthesis . During translation termination, the ribosome remains bound to mRNA with a deacylated tRNA in the P site and an empty A site at the stop codon. RRF and EF-G work cooperatively to catalyze the disassembly of this post-termination complex into its component parts: the 70S ribosome, mRNA, and tRNA .

This recycling step is critical because it allows ribosomes to be reused for subsequent rounds of protein synthesis, preventing ribosomal stalling and maintaining efficient translation throughout the cell. Without functional RRF, ribosomes would remain bound to mRNA after termination, significantly reducing the cell's translational capacity and potentially leading to cell death.

In T. petrophila, as in other bacteria, the RRF is encoded by the frr gene. While experimental data specific to T. petrophila RRF is limited, insights can be gained from the well-characterized RRF of the closely related T. maritima.

How does the structure of RRF relate to its function in ribosome recycling?

The structure of RRF from Thermotoga maritima (a close relative of T. petrophila) reveals a remarkable case of molecular mimicry that explains its functional mechanism. T. maritima RRF consists of two domains: an elongated three-helix bundle domain and a β/β sandwich, forming an L-shaped molecule with dimensions of approximately 70 × 47 × 20 Å .

Given the high sequence similarity between T. maritima and T. petrophila (99.2% 16S rRNA identity), it is reasonable to infer that T. petrophila RRF would adopt a similar structure and mechanism . This structural mimicry represents an elegant evolutionary solution to the problem of ribosome recycling, allowing RRF to interact with the ribosomal binding sites typically occupied by tRNA.

What expression systems are most effective for producing recombinant T. petrophila RRF?

For the expression of recombinant T. petrophila RRF, Escherichia coli-based systems typically provide the most practical approach, with several important considerations:

The primary advantage of expressing thermophilic proteins in mesophilic hosts is the ability to perform a heat treatment purification step. Since T. petrophila has an optimal growth temperature of 80°C, its proteins, including RRF, maintain stability at temperatures that denature most E. coli proteins . A common protocol would involve:

• Cloning the T. petrophila frr gene into a pET vector system with an N-terminal His-tag

• Expression in E. coli BL21(DE3) with IPTG induction at lower temperatures (18-25°C) to improve folding

• Cell lysis followed by heat treatment (65-70°C for 20 minutes) to denature host proteins

• Affinity purification using Ni-NTA chromatography

• Size exclusion chromatography as a final polishing step

When designing the expression construct, codon optimization for E. coli may significantly improve yields, as Thermotoga species have different codon usage patterns. Additionally, including a cleavable fusion partner like SUMO or MBP can enhance solubility, although this is less frequently necessary for intrinsically stable thermophilic proteins.

For analytical-scale expression, researchers should consider testing multiple E. coli strains (BL21, Rosetta, Arctic Express) and induction conditions to optimize protein production before scaling up.

How can researchers reliably assess the activity and stability of recombinant T. petrophila RRF?

Assessing the activity and stability of recombinant T. petrophila RRF requires specialized assays that account for its thermophilic nature:

For activity assessment:

• Ribosome recycling assay: This direct assay measures the ability of RRF to dissociate post-termination complexes in conjunction with EF-G. Researchers can prepare model post-termination complexes using purified ribosomes (either from T. petrophila or from a mesophilic organism like E. coli), then measure complex dissociation through light scattering or sucrose gradient centrifugation.

• EF-G GTPase activation assay: Since RRF stimulates the GTPase activity of EF-G during recycling, GTP hydrolysis can serve as a proxy for RRF function. This can be measured using colorimetric assays for phosphate release or radiolabeled GTP.

• Translational coupling assay: A coupled in vitro translation system can demonstrate RRF activity by showing enhanced translation efficiency in multiple rounds compared to systems lacking functional RRF.

For stability assessment:

• Differential scanning calorimetry (DSC): To determine the melting temperature (Tm) of the protein, with T. petrophila RRF expected to have a Tm significantly higher than mesophilic RRFs

• Circular dichroism spectroscopy: To monitor secondary structure changes during thermal denaturation

• Activity retention after heat treatment: Testing activity after exposure to various temperatures (50-100°C) for defined periods

It is essential to perform these assays at different temperatures, as T. petrophila RRF likely exhibits an activity optimum near the organism's growth temperature (80°C), with potentially different kinetic parameters at mesophilic temperatures.

How does the structure of T. petrophila RRF contribute to its thermostability?

While specific structural data for T. petrophila RRF is not directly provided in the search results, the thermostability mechanisms can be inferred from general principles of protein thermostability in hyperthermophiles:

• Increased ion pairing: Thermophilic proteins typically contain more salt bridges that contribute to structural stability at high temperatures. In T. maritima RRF, and likely T. petrophila RRF, these electrostatic interactions help maintain the relative orientation of the two domains.

• Hydrophobic core optimization: The hydrophobic effect strengthens at higher temperatures, and thermophilic proteins often have more extensive and better packed hydrophobic cores. This would be particularly important for maintaining the structure of the three-helix bundle domain.

• Reduced loop flexibility: Surface loops are often shorter in thermophilic proteins to reduce entropy-driven unfolding. The L-shaped structure of RRF, with its tRNA-mimicking conformation, would benefit from rigid connecting regions between domains.

• Secondary structure stabilization: Both the α-helical bundle and β/β sandwich domains in RRF would likely contain additional hydrogen bonds and salt bridges in the thermophilic version compared to mesophilic homologs.

• Surface charge optimization: Thermophilic proteins often display increased surface charge density, which can enhance stability through extensive electrostatic networks.

Comparative structural analysis between T. petrophila RRF and mesophilic RRFs would reveal these adaptations in detail. Such information is valuable not only for understanding thermoadaptation but also for protein engineering applications seeking to enhance the stability of other proteins.

What are the optimal buffer conditions for maintaining stability and activity of recombinant T. petrophila RRF?

The optimal buffer conditions for T. petrophila RRF must account for both stability at high temperatures and maintaining native conformation. Based on principles for handling thermophilic proteins, recommended buffer considerations include:

• Buffer type: Phosphate buffers provide excellent thermal stability and minimal pH changes with temperature variation. Alternatively, HEPES or MOPS buffers with appropriate pKa adjustment for high-temperature applications can be used.

• pH optimization: The optimal pH should be determined experimentally, but typically falls between 7.0-8.0 for RRF proteins. It's important to account for pH changes with temperature (ΔpKa/ΔT), which can be significant when working at elevated temperatures.

• Salt concentration: Moderate to high salt concentrations (100-300 mM NaCl) often enhance thermostable protein stability by strengthening hydrophobic interactions.

• Stabilizing additives:

  • Glycerol (5-10%) to prevent aggregation during freeze-thaw cycles

  • Divalent cations (especially Mg²⁺, 5-10 mM) which are often required for RRF function

  • Reducing agents (DTT or TCEP) to prevent oxidation of cysteine residues

• Storage conditions: For long-term storage, flash freezing in liquid nitrogen and storage at -80°C in small aliquots to avoid repeated freeze-thaw cycles

For activity assays specifically, the buffer should include components necessary for RRF function:

• Mg²⁺ (typically 5-10 mM) for ribosome binding

• Monovalent cations (K⁺ or NH₄⁺, 50-100 mM) to maintain ionic strength

• GTP (0.5-1 mM) for EF-G function in coupled assays

When determining optimal conditions, researchers should test stability and activity across various buffer compositions and temperatures ranging from 60-90°C to find the optimal working conditions for T. petrophila RRF.

What site-directed mutagenesis approaches can be used to investigate the structure-function relationship of T. petrophila RRF?

Site-directed mutagenesis provides valuable insights into the structure-function relationship of T. petrophila RRF. Based on what is known about RRF structure, particularly from T. maritima, several targeted approaches are recommended:

• Domain interface mutations: Since RRF consists of two domains that form an L-shaped molecule mimicking tRNA , mutations at the domain interface could reveal the importance of domain orientation and flexibility. Key residues at this interface could be mutated to determine if domain movement is required during the recycling process.

• Ribosome binding residues: Mutations in conserved surface residues likely involved in ribosome binding, particularly those in the three-helix bundle domain, would help map the critical interaction surfaces. Progressive alanine scanning of surface patches can identify essential binding regions.

• EF-G interaction sites: Residues involved in EF-G coordination should be identified and mutated to understand the cooperative mechanism between these factors. This is particularly important as the synergy between RRF and EF-G is essential for ribosome recycling.

• Thermostability determinants: Comparative sequence analysis between T. petrophila RRF and mesophilic RRFs can identify unique residues that may contribute to thermostability. These could include:

  • Additional charged residues forming salt bridges

  • Proline residues in loops that restrict flexibility

  • Hydrophobic residues in the protein core

• Functional complementation testing: Mutants should be tested in a complementation assay using an E. coli temperature-sensitive frr mutant to assess in vivo functionality.

Each mutation should be carefully designed based on sequence conservation analysis and structural modeling. The resulting mutant proteins should be characterized for:

• Stability (using thermal denaturation assays)

• Ribosome binding affinity (using surface plasmon resonance)

• Functional activity (using ribosome recycling assays)

• EF-G interaction (using pull-down assays or isothermal titration calorimetry)

This systematic approach will generate a comprehensive map of structure-function relationships in T. petrophila RRF.

How does T. petrophila RRF differ structurally and functionally from RRFs in mesophilic bacteria?

The structural and functional differences between T. petrophila RRF and mesophilic RRFs reflect adaptations to their respective thermal environments:

Structurally, while the search results don't provide direct comparative data, we can infer that T. petrophila RRF would share the tRNA-mimicking L-shaped conformation observed in T. maritima RRF , but with several thermoadaptive features that distinguish it from mesophilic homologs:

• Increased rigidity: T. petrophila RRF likely has a more rigid structure at room temperature compared to mesophilic RRFs, with comparable flexibility only at its physiological temperature (80°C) .

• Electrostatic network: Enhanced surface ion-pairing networks would stabilize the structure at high temperatures, often visible as clusters of charged residues on the protein surface.

• Compactness: The hydrophobic core is likely more tightly packed, with fewer and smaller internal cavities compared to mesophilic RRFs.

Functionally, key differences would include:

• Temperature optima: T. petrophila RRF would function optimally at temperatures around 80°C , whereas mesophilic RRFs typically denature at such temperatures.

• Kinetic parameters: At low temperatures (20-37°C), T. petrophila RRF might show reduced activity compared to mesophilic RRFs, but would maintain activity at temperatures where mesophilic proteins denature.

• Conformational dynamics: The structural changes required for function likely require higher activation energy in T. petrophila RRF, consistent with the need for molecular rigidity at high temperatures.

• Interaction with EF-G: The interaction interface between T. petrophila RRF and its cognate EF-G may contain additional stabilizing features to maintain functional coupling at elevated temperatures.

These differences highlight evolutionary adaptations that allow T. petrophila RRF to maintain the same fundamental function as its mesophilic counterparts but in an extreme thermal environment.

What insights can be gained from studying the mannooligosaccharide transport systems of T. petrophila for understanding its broader metabolic adaptations?

While not directly related to RRF function, the search results provide interesting comparative data on mannooligosaccharide transport systems in Thermotoga species that illustrate broader metabolic adaptations:

T. petrophila shows significant upregulation of mannooligosaccharide utilization genes when grown on polysaccharide mixtures compared to glucose . The following table illustrates differential gene expression in Thermotoga species related to mannan utilization:

This data reveals several important points about T. petrophila metabolism:

These metabolic differences highlight how even closely related Thermotoga species have fine-tuned their carbohydrate utilization systems through evolution, likely reflecting adaptations to their specific habitats. Similar species-specific adaptations may exist in core cellular processes including translation and ribosome recycling, which could potentially influence the functional properties of T. petrophila RRF.

What crystallization approaches would be most suitable for solving the structure of T. petrophila RRF?

For determining the crystal structure of T. petrophila RRF, researchers should consider approaches that leverage its inherent thermostability:

• Initial screening considerations:

  • Traditional sparse matrix screens at both room temperature and 4°C

  • Higher protein concentrations (10-20 mg/ml) than typically used for mesophilic proteins

  • Inclusion of additives that promote crystal packing, such as low percentages of polyethylene glycols or alcohols

• Thermophile-specific strategies:

  • In situ crystallization at elevated temperatures (40-60°C) using temperature-controlled crystallization plates

  • Exploration of higher salt concentrations (0.5-2.0 M) that may better mimic the intracellular ionic strength of T. petrophila

  • Co-crystallization with stabilizing ligands or binding partners (like fragments of EF-G)

• Data collection approach:

  • Multi-wavelength anomalous dispersion (MAD) using selenomethionine-substituted protein, as was successfully employed for T. maritima RRF

  • Collection at cryogenic temperatures following careful optimization of cryoprotectant conditions

• Alternative approaches if crystallization proves challenging:

  • Surface entropy reduction mutations to promote crystal contacts

  • Truncation constructs guided by limited proteolysis experiments

  • Crystallization in complex with binding partners or antibody fragments

The search results indicate that T. maritima RRF was successfully crystallized and its structure determined using selenomethionine substitution and MAD phasing to 2.9 Å resolution at beamline BM14 . Given the high sequence similarity between T. maritima and T. petrophila, similar crystallization conditions might be successful, with data collection at a synchrotron facility providing the best chance of obtaining high-resolution diffraction data.

How might T. petrophila RRF be engineered for biotechnological applications?

The extreme thermostability and specific functional properties of T. petrophila RRF present several opportunities for biotechnological applications:

• Thermostable expression tag: The inherent stability of T. petrophila RRF could be exploited as a fusion tag to enhance the expression, solubility, and thermostability of other recombinant proteins. This would be particularly valuable for industrial enzymes requiring high-temperature applications.

• Ribosome engineering: Understanding the interaction between T. petrophila RRF and ribosomes could inform the development of engineered translation systems with enhanced properties, such as:

  • Increased tolerance to organic solvents

  • Ability to function at elevated temperatures for cell-free protein synthesis

  • Resistance to translation inhibitors

• Chimeric RRFs: Creating chimeric proteins combining domains from T. petrophila RRF and mesophilic RRFs could produce variants with tailored stability and activity profiles for specific biotechnological applications.

• Structure-guided stability engineering: The thermostability principles identified in T. petrophila RRF could be applied to engineer enhanced stability in other translation factors for various biotechnological applications, including:

  • Cell-free protein synthesis systems

  • Protein production in extremophilic hosts

  • Development of heat-resistant microbial cell factories

• Antimicrobial development: The structural differences between bacterial RRFs (including T. petrophila RRF) and the absence of RRF in eukaryotes make it a potential target for novel antimicrobial development. Structural insights from thermostable RRFs could inform rational drug design targeting the bacterial protein synthesis machinery.

To pursue these applications, researchers would need to thoroughly characterize T. petrophila RRF's structure-function relationships and develop effective expression and engineering platforms tailored to this thermostable protein.