Recombinant Geobacillus thermodenitrificans Peptide chain release factor 1 (prfA)

What is Peptide chain release factor 1 (prfA) and what is its function in Geobacillus thermodenitrificans?

Peptide chain release factor 1 (prfA) in G. thermodenitrificans is a protein involved in translation termination that recognizes the stop codons UAA and UAG, catalyzing the release of completed polypeptide chains from the ribosome. Similar to the mechanism observed in Bacillus subtilis, prfA is likely essential for proper protein synthesis termination in G. thermodenitrificans .

The functionality of prfA must be understood within the context of G. thermodenitrificans as a thermophilic organism with optimal growth at 70°C. The thermostability of its proteins, including prfA, represents an adaptation to high-temperature environments such as deep oil reservoirs where this bacterium has been isolated . The protein likely contains structural elements that contribute to heat resistance while maintaining catalytic activity at elevated temperatures.

How does the G. thermodenitrificans genome compare to other Geobacillus species?

The G. thermodenitrificans K1041 genome consists of 3,755,826 bp with a GC content of 49.18% and contains 3,848 genes, including 3,608 protein genes, 117 pseudogenes, 88 tRNAs, 30 rRNAs, and 5 ncRNAs . This is similar in size to the genome of G. kaustophilus HTA426, which contains a 3,544,776-bp chromosome and a 47,890-bp plasmid .

Another strain, G. thermodenitrificans NG80-2, isolated from a deep oil reservoir in Northern China, has a 3,550,319-bp chromosome and a 57,693-bp plasmid with mean G+C contents of 49.0% and 39.8%, respectively . This strain contains 3,499 predicted ORFs, 11 rRNA operons, and 87 tRNA genes, covering 86% of its genome .

The comparison below highlights key genomic features across different Geobacillus strains:

What are the optimal expression systems for recombinant production of G. thermodenitrificans prfA?

For recombinant expression of G. thermodenitrificans prfA, Escherichia coli remains the most commonly used heterologous host due to its ease of genetic manipulation and rapid growth. A methodology similar to that used for recombinant glutaminase from G. thermodenitrificans DSM-465 could be applied . The recombinant glutaminase was successfully expressed in E. coli with 40% recovery and 22.36-fold purity following purification to electrophoretic homogeneity .

For thermostable proteins like prfA, expression systems that can properly fold these proteins are critical. Consider the following host systems:

• E. coli BL21(DE3): Most commonly used, particularly with pET vectors under T7 promoter control

• E. coli Rosetta: Enhanced expression of proteins containing rare codons

• Bacillus subtilis: As a Gram-positive host more closely related to Geobacillus

• Geobacillus itself: Some strains like G. thermodenitrificans K1041 and T12 can be efficiently transformed via electroporation

Expression variables to optimize include:

• Induction temperature (lowering to 18-25°C may improve folding)

• IPTG concentration (typically 0.1-1.0 mM)

• Duration of expression (4-24 hours)

• Media composition (LB, TB, or auto-induction media)

How do the structural features of G. thermodenitrificans prfA contribute to its thermostability?

While specific structural information about G. thermodenitrificans prfA is limited, thermostable proteins generally exhibit several characteristic features that likely apply to prfA:

• Increased internal hydrophobicity: More extensive hydrophobic core interactions

• Higher proportion of charged residues: Enhanced ionic interactions at the protein surface

• Decreased loop regions: Reduced flexibility in non-structured regions

• Increased proline content: Greater conformational rigidity

• More extensive hydrogen bonding and salt bridge networks: Enhanced structural stability

Molecular modeling approaches similar to those used for glutaminase could elucidate the structural basis of thermostability in prfA. Despite having less than 40% amino acid identity with human homologs, the G. thermodenitrificans glutaminase exhibited ∼94% structural conservation in key domains, suggesting a similar pattern might exist for prfA .

What are the molecular mechanisms of stop codon recognition by G. thermodenitrificans prfA?

The stop codon recognition mechanism by G. thermodenitrificans prfA likely shares similarities with other bacterial release factors, with species-specific adaptations for thermostability. In bacteria, RF1 typically recognizes UAA and UAG stop codons while RF2 recognizes UAA and UGA.

The selectivity in codon recognition depends on a tripeptide sequence within the release factor that interacts with the stop codon in the ribosomal A site. For most bacterial RF1 proteins, this sequence is Pro-X-Thr, where X is a variable amino acid. This mechanism would likely be conserved in G. thermodenitrificans prfA.

The molecular basis of this recognition would involve:

• Binding of prfA to the ribosomal A site

• Recognition of the stop codon by the tripeptide motif

• Conformational changes in prfA to position its catalytic GGQ motif

• Hydrolysis of the ester bond linking the peptide to the tRNA

These mechanisms could be investigated using cryo-EM structures of prfA bound to ribosomes, similar to the approach used to characterize BrfA, a ribosome rescue factor in Bacillus subtilis . BrfA was shown to bind to stalled ribosomes and recruit RF2, inducing a transition to an open active conformation . This methodology could be adapted to study prfA interactions.

How does G. thermodenitrificans manage ribosome rescue compared to other bacteria?

Ribosome rescue is essential for bacterial survival when translation stalls on non-stop mRNAs. While most bacteria use trans-translation mediated by tmRNA and SmpB, some have evolved alternative rescue pathways.

In Bacillus subtilis, a Gram-positive bacterium related to Geobacillus, a ribosome rescue factor named BrfA has been identified. BrfA binds to non-stop stalled ribosomes and recruits RF2 (but not RF1), inducing a conformational change that enables RF2 to catalyze peptide release despite the absence of a stop codon .

G. thermodenitrificans likely possesses similar rescue mechanisms, either through:

• The canonical trans-translation system

• A BrfA-like alternative rescue factor

• A combination of both systems for redundancy

Genetic analysis in B. subtilis has shown that either trans-translation or BrfA is required for growth, even without additional stress . This suggests that G. thermodenitrificans would also require efficient ribosome rescue mechanisms, particularly considering the additional stress of high-temperature environments.

Research into G. thermodenitrificans ribosome rescue would provide insights into how thermophilic bacteria maintain protein synthesis fidelity under thermal stress conditions and could reveal thermostable variants of rescue factors with biotechnological applications.

What are the best methods for purifying recombinant G. thermodenitrificans prfA while maintaining its activity?

Purification of recombinant G. thermodenitrificans prfA requires protocols that preserve both structure and function. Based on successful approaches with other thermostable proteins from G. thermodenitrificans, the following protocol is recommended:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

• Heat treatment: Incubation at 60-65°C for 15-20 minutes to denature E. coli proteins while retaining thermostable prfA

• Affinity chromatography: Using either:

  • His-tag purification with Ni-NTA resin

  • GST-tag purification if solubility is an issue

• Ion exchange chromatography: Q-Sepharose or SP-Sepharose depending on prfA pI

• Size exclusion chromatography: Final polishing step using Superdex 75/200

This multi-step approach has shown success with G. thermodenitrificans glutaminase, achieving 40% recovery and 22.36-fold purity . The heat treatment step is particularly advantageous when purifying thermostable proteins from mesophilic expression hosts.

Storage in buffer containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl₂, 5% glycerol, and 1 mM DTT at -80°C is recommended to maintain activity. Avoid multiple freeze-thaw cycles.

How can we assess the functionality of recombinant G. thermodenitrificans prfA in vitro?

In vitro assessment of recombinant G. thermodenitrificans prfA functionality requires assays that measure its peptide release activity. The following methodologies are recommended:

1. Ribosome-dependent peptidyl-tRNA hydrolysis assay:

• Prepare pre-termination complexes (pre-TCs) with ribosomes, mRNA containing a stop codon, and peptidyl-tRNA

• Add purified prfA

• Measure the release of the peptide using either:

  • Radiolabeled peptides and quantification via scintillation counting

  • Fluorescently labeled peptides and quantification via fluorescence spectroscopy

2. Dual-luciferase reporter assay:

• Construct a bicistronic reporter with firefly and Renilla luciferase separated by a stop codon

• Express in a cell-free translation system supplemented with purified prfA

• Measure the ratio of firefly to Renilla luciferase activity

3. In vitro translation termination efficiency assay:

• Set up in vitro translation reactions with purified components

• Use mRNAs with different stop codons (UAA, UAG, UGA)

• Quantify the amount of full-length protein produced versus truncated products

4. Temperature-dependent activity profile:

• Perform the above assays at different temperatures (30-80°C)

• Determine the optimal temperature and thermostability profile

• Compare with mesophilic release factors to highlight thermoadaptations

For kinetic parameters, determine the KM and kcat using varying concentrations of pre-TCs or synthetic substrates. The approach used for G. thermodenitrificans glutaminase determined a KM value of 104 μM for L-glutamine , and similar methods could be applied to prfA using appropriate substrates.

What in silico approaches can predict interactions between G. thermodenitrificans prfA and potential inhibitors?

Computational approaches provide valuable insights into protein-ligand interactions before experimental validation. For studying G. thermodenitrificans prfA interactions, the following in silico workflow is recommended:

1. Homology modeling:

• Build a 3D model using Swiss-Model or similar tools

• Use known bacterial RF1 structures as templates

• Validate the model using PROCHECK, VERIFY3D, and ProSA

2. Molecular dynamics simulations:

• Perform temperature-dependent simulations (60-80°C)

• Analyze conformational stability and flexibility

• Identify potential binding pockets that exist at elevated temperatures

3. Molecular docking:

• Screen potential inhibitors against identified binding pockets

• Calculate binding energies and rank compounds

• Analyze binding modes and key interactions

4. Comparative analysis with homologous proteins:

• Use ConSurf server to identify evolutionarily conserved residues

• Apply TM-align for structural comparisons with other release factors

• Identify unique features of G. thermodenitrificans prfA

This approach was successfully used for G. thermodenitrificans glutaminase, where molecular docking identified CB-839 as the best inhibitor with a binding free energy change (ΔG) of -388.7 kJ mol⁻¹ . A similar approach could rank potential prfA inhibitors based on binding energies.

For advanced analysis, molecular dynamics simulations at elevated temperatures (60-80°C) can reveal thermostability mechanisms and temperature-dependent conformational changes relevant to prfA function in thermophilic environments.

How can G. thermodenitrificans prfA be engineered for enhanced thermostability and activity?

Engineering G. thermodenitrificans prfA for enhanced properties requires a combination of rational design and directed evolution approaches:

Rational design strategies:

• Consensus-based mutations: Align prfA sequences from hyperthermophiles and introduce consensus residues

• Disulfide bridge engineering: Introduce non-native disulfide bonds to stabilize flexible regions

• Surface charge optimization: Increase surface charged residues to enhance ionic interactions

• Loop stabilization: Shorten or rigidify flexible loop regions

Directed evolution methods:

• Error-prone PCR: Generate random mutations followed by screening at extreme temperatures

• DNA shuffling: Recombine prfA genes from different thermophilic species

• PACE (Phage-Assisted Continuous Evolution): Adapt for continuous evolution of thermostability

• Deep mutational scanning: Systematically assess all possible single mutations

The success of these approaches can be measured using thermal shift assays (Thermofluor), differential scanning calorimetry, and activity assays at increasing temperatures. Engineering efforts should focus on maintaining the critical catalytic residues while modifying the surrounding structure for enhanced stability.

Comparative analysis with other thermostable proteins from G. thermodenitrificans, such as its glutaminase which shows remarkable structural conservation despite sequence divergence, could provide insights into permissible modification sites .

What role might G. thermodenitrificans prfA play in maintaining protein synthesis at elevated temperatures?

Thermophilic organisms face unique challenges in maintaining protein synthesis at elevated temperatures, including increased risk of translational errors and protein misfolding. G. thermodenitrificans prfA likely plays several critical roles in this context:

• Efficient translation termination: Preventing readthrough errors that could produce extended proteins with aberrant functions

• Ribosome quality control: Participating in rescue of stalled ribosomes, similar to the BrfA system in B. subtilis

• Preventing ribosome damage: Efficient release of completed peptides to avoid ribosome sequestration

• Maintaining translation rates: Ensuring optimal protein synthesis speed despite thermal stress

The genome of G. thermodenitrificans NG80-2 reveals adaptations for surviving in fluctuating environments, including genes for various transporters, detoxification systems, and flexible respiration systems . Similar adaptations likely exist in the translation machinery, with prfA being a key component.

Future research directions should include:

• Comparative analysis of translation termination efficiency between thermophilic and mesophilic release factors

• Investigation of potential thermophile-specific post-translational modifications of prfA

• Examination of prfA interactions with other translation factors at elevated temperatures

• Analysis of prfA expression levels under different stress conditions

How does G. thermodenitrificans prfA compare to release factors from pathogenic bacteria as a potential antibiotic target?

While G. thermodenitrificans itself is not pathogenic, comparing its prfA to release factors from pathogenic bacteria provides insights into potential antibiotic development:

Structural comparisons:

• Despite sequence divergence, bacterial release factors often maintain high structural similarity

• The G. thermodenitrificans glutaminase showed ~94% structural conservation with human homologs despite <40% sequence identity

• Similar conservation patterns might exist for prfA, making it a valuable model for studying release factor inhibition

Potential advantages as a drug development model:

• Thermostability: Facilitates structural studies and assay development

• Non-pathogenic source: Allows safer handling during initial inhibitor screening

• Evolutionary insights: Reveals conserved features essential for function across bacterial species

Inhibitor development strategy:

• Perform molecular docking with potential inhibitors, as done for G. thermodenitrificans glutaminase

• Target conserved active site residues shared with pathogenic species

• Design inhibitors that can discriminate between bacterial and human release factors

• Test promising compounds against both thermophilic and mesophilic release factors

The following table compares features relevant to antibiotic development:

What are common challenges in heterologous expression of G. thermodenitrificans prfA and how can they be addressed?

Heterologous expression of thermophilic proteins like G. thermodenitrificans prfA presents several challenges:

1. Protein misfolding and inclusion body formation:

• Solution: Lower induction temperature (16-25°C), use solubility-enhancing fusion tags (SUMO, MBP), or add osmolytes (sorbitol, glycine betaine)

• Alternative approach: Refold from inclusion bodies using optimized protocols for thermostable proteins

2. Codon usage bias:

• Solution: Use codon-optimized synthetic genes or expression hosts with rare tRNA genes (e.g., E. coli Rosetta)

• Analysis method: Calculate codon adaptation index (CAI) to identify problematic regions

3. Toxicity to host cells:

• Solution: Use tightly regulated expression systems (e.g., pET with T7-lysozyme) or secretion-based expression

• Alternative hosts: Consider Bacillus subtilis or other Gram-positive expression systems

4. Post-translational modifications:

• Solution: Use eukaryotic expression systems if modifications are essential for function

• Analysis: Perform mass spectrometry to identify any modifications present in native prfA

5. Low yield:

• Solution: Optimize media composition, inducer concentration, and harvest time

• Scale-up: Consider high-density fermentation or auto-induction media

G. thermodenitrificans K1041 and T12 strains have been identified as efficient recipients for transformation via electroporation, which is uncommon in G. thermodenitrificans . This suggests potential for homologous expression of prfA, which could overcome many of the challenges associated with heterologous expression.

How can contradictory experimental results in G. thermodenitrificans prfA research be reconciled?

When faced with contradictory experimental results in prfA research, apply the following systematic approach:

1. Experimental conditions analysis:

• Temperature effects: Results may differ significantly between standard lab temperatures (37°C) and the optimal temperature for G. thermodenitrificans (60-70°C)

• Buffer composition: Ionic strength and pH can dramatically affect thermostable protein behavior

• Protein concentration: Self-association or different oligomeric states at varying concentrations

2. Construct design differences:

• Tag position and type: N-terminal vs. C-terminal tags may differently affect function

• Linker sequences: Rigid vs. flexible linkers between domains or tags

• Truncation effects: Different functional domains included or excluded

3. Statistical validation:

• Sample size evaluation: Ensure sufficient replicates for statistical significance

• Outlier analysis: Apply appropriate statistical tests to identify and address outliers

• Methodological biases: Consider systematic errors inherent to different techniques

4. Source strain variation:

• G. thermodenitrificans strain differences: Compare genomes of different strains (NG80-2, K1041, DSM-465)

• Evolutionary analysis: Reconcile differences through phylogenetic analysis

A useful approach for resolving contradictions is to apply multiple complementary techniques to the same question, similar to the approach used in search result 8, where biochemical characterization was combined with cryo-EM structural analysis to understand the function of the ribosome rescue factor BrfA .

Computational tools for detecting contradictions in scientific literature, such as those described in search result 3, could also be applied to systematically identify and categorize contradictory findings about G. thermodenitrificans proteins .

How can expertise from different fields contribute to G. thermodenitrificans prfA research?

G. thermodenitrificans prfA research offers rich opportunities for interdisciplinary collaboration:

1. Structural biology and biophysics:

• X-ray crystallography or cryo-EM to determine prfA structure

• Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• NMR studies to analyze domain movements during substrate binding

2. Computational biology:

• Molecular dynamics simulations at elevated temperatures

• Quantum mechanics/molecular mechanics (QM/MM) to model catalytic mechanisms

• Machine learning approaches to predict stability-enhancing mutations

3. Microbiology and genetics:

• Development of genetic tools for G. thermodenitrificans

• Creation of conditional knockdowns to study prfA essentiality

• Transcriptomic analysis of translation-related genes under stress

4. Biochemistry and biophysics:

• Enzyme kinetics at various temperatures

• Thermodynamic analysis of protein stability

• Single-molecule studies of prfA-ribosome interactions

5. Synthetic biology:

• Engineering of thermostable translation systems

• Development of cell-free protein synthesis platforms using thermostable components

• Creation of minimal translation systems with defined components

Collaborative research could follow the approach of the Aligning Science Across Parkinson's (ASAP) initiative described in search result 7, which emphasizes interdisciplinary collaboration focused on specific scientific themes . A similar model could be applied to thermophilic translation termination research, bringing together experts from structural biology, microbiology, and biochemistry.

What are promising future research directions for G. thermodenitrificans prfA?

Future research on G. thermodenitrificans prfA should explore several promising directions:

1. Structural biology:

• High-resolution structures of prfA in different functional states

• Cryo-EM structures of prfA bound to ribosomes at termination codons

• Comparison with mesophilic homologs to identify thermoadaptations

2. Biotechnological applications:

• Development of thermostable cell-free protein synthesis systems

• Engineering prfA for specific stop codon suppression applications

• Creating chimeric release factors with novel specificities

3. Evolutionary biology:

• Comparative analysis of release factors across the Geobacillus genus

• Investigation of horizontal gene transfer events involving translation factors

• Reconstruction of ancestral release factor sequences to trace thermoadaptation

4. Systems biology:

• Global analysis of translation termination efficiency at high temperatures

• Integration of prfA function with other aspects of the heat shock response

• Metabolic consequences of translation termination errors

5. Synthetic biology:

• Engineering minimal thermostable translation systems

• Development of orthogonal translation systems for incorporating non-standard amino acids

• Creation of temperature-responsive gene expression systems

These research directions align with broader initiatives in understanding bacterial adaptation to extreme environments and could contribute to the development of novel biotechnological applications leveraging the thermostability of G. thermodenitrificans proteins.