Recombinant Photobacterium profundum Ribosome-recycling factor (frr)

What is the function of Ribosome-recycling factor (frr) in Photobacterium profundum?

Ribosome-recycling factor (frr) in Photobacterium profundum, like in other bacteria, plays a critical role in the fourth and final stage of protein synthesis. After termination, frr works in concert with elongation factor G (EF-G) and GTP to catalyze the disassembly of the post-termination complex (PoTC). This complex comprises the 70S ribosome with messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon. The recycling process involves breaking down this complex to release mRNA and tRNA from the ribosome and splitting the ribosome into its 30S and 50S subunits, making these components available for subsequent rounds of protein synthesis .

In marine bacteria such as P. profundum, which can thrive under various pressure conditions, ribosome recycling may have adapted to function efficiently across different environmental pressures. The genome of P. profundum 3TCK, a shallow bathytype strain, has been sequenced and compared with the deep bathytype strain SS9, revealing genomic adaptations to different depth environments .

What are the optimal expression systems for producing recombinant P. profundum frr?

Multiple expression systems can be employed for the production of recombinant P. profundum Ribosome-recycling factor, each with distinct advantages:

E. coli and yeast expression systems offer the highest yields and shorter production times, making them particularly suitable for structural and basic biochemical studies . For studies requiring proper post-translational modifications that may be necessary for correct protein folding or optimal activity, insect cells with baculovirus or mammalian expression systems are recommended, despite their lower yields and longer production times .

How does P. profundum frr structure compare to frr from other bacterial species?

While the search results do not provide specific structural information about P. profundum frr, bacterial ribosome recycling factors are generally conserved in structure and function across species. Based on the genomic features of P. profundum strains, which have a GC content of approximately 41.3% , we might expect some amino acid composition differences compared to other bacterial frr proteins.

The structural features likely include a domain that mimics tRNA to facilitate interaction with the ribosome, as observed in other bacterial species. Given that P. profundum exists in different ecotypes adapted to varying pressures (shallow bathytype 3TCK versus deep bathytype SS9), researchers should consider potential structural adaptations that might enable function under different pressure conditions.

What purification strategy yields the highest activity of recombinant P. profundum frr?

For optimal purification of recombinant P. profundum frr with maximal activity, a multi-step chromatography approach is recommended:

• Initial capture using affinity chromatography (such as His-tag or GST-tag systems) depending on the fusion protein design

• Intermediate purification via ion exchange chromatography, optimized for the specific pI of P. profundum frr

• Polishing step using size exclusion chromatography to ensure homogeneity and remove aggregates

When expressing P. profundum frr in E. coli systems, which offer high yields and short turnaround times , inclusion body formation may occur. In such cases, optimized refolding protocols using gradual dialysis against decreasing concentrations of denaturants while maintaining reducing conditions have proven effective for recovering active protein.

For applications requiring highly active protein, expression in insect cells with baculovirus may provide the necessary post-translational modifications for correct protein folding and retention of activity . In this case, purification should be performed under gentler conditions to preserve the native structure.

How can researchers design kinetic assays to measure P. profundum frr activity in vitro?

To assess the activity of recombinant P. profundum frr in vitro, researchers can implement several complementary assays that measure different aspects of the ribosome recycling process:

• Ribosome Splitting Assay: Monitor the conversion of 70S ribosomes to 30S and 50S subunits using light scattering or sucrose gradient centrifugation. The reaction mixture should contain purified P. profundum frr, EF-G, GTP, and post-termination complexes assembled from P. profundum components if available, or from a model organism .

• mRNA Release Assay: Utilize fluorescently labeled mRNA to track its release from the post-termination complex in real-time. This can be measured by changes in fluorescence anisotropy or by filter-binding assays quantifying the amount of released mRNA versus ribosome-bound mRNA .

• tRNA Release Assay: Similar to the mRNA release assay, use fluorescently labeled tRNA to monitor its dissociation from the ribosome, providing insights into the kinetic mechanism of recycling .

When designing these assays, it's crucial to consider the possible effects of environmental factors such as pressure and salt concentration, as P. profundum is a marine bacterium that exists in different ecotypes adapted to varying depths .

What controls are essential when evaluating the functional complementation of frr in bacterial systems?

When conducting functional complementation studies with P. profundum frr in bacterial systems, the following controls are essential:

• Positive Control: Wild-type frr from the host organism (e.g., E. coli frr when using an E. coli system) to establish baseline complementation efficiency.

• Negative Controls:

  • Empty vector control to confirm that observed effects are due to the expressed protein

  • Catalytically inactive mutant of P. profundum frr (e.g., with mutations in key residues involved in GTP hydrolysis or ribosome binding)

• Environmental Controls: Since P. profundum exists as different ecotypes adapted to varying pressures , complementation experiments should be conducted under different pressure conditions to evaluate pressure-dependent functionality.

• Depletion Control: A system where endogenous frr can be conditionally depleted, similar to the RRF knockdown (KD) system described for E. coli , to confirm that the recombinant P. profundum frr can rescue the depletion phenotype.

These controls ensure that any observed complementation is specifically due to the functional activity of the recombinant P. profundum frr and not to experimental artifacts or residual activity of endogenous proteins.

How does hydrostatic pressure affect the kinetics of P. profundum frr-mediated ribosome recycling?

The effect of hydrostatic pressure on P. profundum frr activity represents a crucial area of investigation, given the adaptation of different P. profundum ecotypes to varying ocean depths . Research approaches to address this question should include:

• Comparative Kinetic Analysis: Measure the rates of ribosome splitting, mRNA release, and tRNA release at different pressures (from atmospheric to deep-sea pressures of approximately 30 MPa) using the assays described in section 2.2. Compare the kinetic parameters (kcat, KM) of frr from shallow bathytype 3TCK versus deep bathytype SS9 strains .

• Structural Stability Assessment: Evaluate the structural integrity and stability of recombinant frr proteins from different P. profundum ecotypes under varying pressures using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (HDX-MS).

• Molecular Dynamics Simulations: Perform computational analyses to predict pressure-induced conformational changes in frr structure and how these might impact interactions with ribosomes, EF-G, and GTP.

Preliminary data suggest that deep-sea adapted proteins often contain specific amino acid substitutions that confer barotolerance. The complete genome sequences of P. profundum strains reveal adaptations to different depth environments , which likely extend to functional modifications in translational machinery proteins including frr.

What is the impact of frr depletion on translational coupling in P. profundum operons?

The impact of frr depletion on translational coupling in P. profundum operons represents an intriguing research question, particularly in light of findings from E. coli studies:

Recent research in E. coli demonstrated that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide, suggesting that re-initiation is not a major mechanism of translational coupling in E. coli . To investigate whether this holds true for P. profundum, researchers should:

• Develop a Conditional Depletion System: Create a system for conditional depletion of frr in P. profundum, similar to the RRF knockdown strategy used in E. coli .

• Ribosome Profiling Analysis: Perform ribosome profiling before and after frr depletion to analyze ribosome distribution across genes within polycistronic messages. This approach can reveal whether frr depletion alters the ratio of ribosome density on neighboring genes within operons .

• Reporter Assays: Design bicistronic reporter constructs containing pairs of genes from P. profundum operons to directly measure coupling efficiency with and without frr depletion .

• High-Salt Ribosome Profiling: Implement high-salt ribosome profiling to differentiate between elongating ribosomes and post-termination complexes, as was done in E. coli studies .

The genome organization of P. profundum into two chromosomes with specific gene arrangements may influence translational coupling mechanisms in ways that differ from E. coli, making this comparison particularly valuable.

How do the kinetic mechanisms of ribosome recycling differ between shallow and deep ecotypes of P. profundum?

The kinetic mechanisms of ribosome recycling may differ substantially between shallow bathytype (e.g., 3TCK) and deep bathytype (e.g., SS9) strains of P. profundum due to adaptations to their respective environmental pressures :

• Order of Events Analysis: Determine whether the sequence of events in ribosome recycling (mRNA release, tRNA release, and 70S splitting) occurs in the same order in both ecotypes under their native pressure conditions. In common bacterial post-termination complexes, recycling typically proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting .

• Rate-Limiting Step Identification: Identify the rate-limiting step in the recycling process for each ecotype through pre-steady-state kinetic measurements. Compare whether frr from deep bathytype strains has evolved to overcome potential pressure-induced constraints on conformational changes required for recycling.

• Interaction Analysis with Partner Proteins: Investigate whether frr from different ecotypes exhibits altered binding affinities or interaction kinetics with EF-G and ribosomes under varying pressure conditions using surface plasmon resonance (SPR) or bio-layer interferometry (BLI).

Comparison of frr sequences and structures from these ecotypes, in conjunction with functional assays, would provide insights into the molecular adaptations that enable efficient ribosome recycling across different environmental pressures, contributing to our understanding of how essential cellular processes adapt to extreme conditions.

How can researchers resolve contradictory data regarding the role of P. profundum frr in ribosome subunit splitting versus mRNA/tRNA release?

Resolving contradictory data regarding the primary role of P. profundum frr—whether it primarily facilitates ribosome subunit splitting or mRNA/tRNA release—requires a comprehensive experimental approach:

• Temporal Resolution Experiments: Implement time-resolved experiments that can simultaneously track multiple events during recycling. This might involve using different fluorescent labels for mRNA, tRNA, and ribosomal subunits, allowing researchers to determine the precise order and kinetics of each step .

• Mutational Analysis: Generate and characterize frr mutants with selective defects in specific aspects of recycling. Mutants that can promote mRNA/tRNA release but not subunit splitting, or vice versa, would help delineate the primary function.

• Cross-Species Complementation: Assess whether frr from P. profundum can complement RRF-depleted strains of other bacteria, such as E. coli, and whether the pattern of 3′-UTR ribosome accumulation and translational coupling matches that of the native RRF or shows distinct characteristics .

The controversy in bacterial ribosome recycling centers on whether RRF and EF-G·GTP primarily effect the release of mRNA and tRNA from the ribosome, with subunit splitting being somewhat dispensable, or whether their main function is to catalyze the splitting reaction . Systematically designed experiments that address both possibilities in the specific context of P. profundum could resolve these contradictions.

What explains variability in recombinant P. profundum frr activity when expressed in different host systems?

Variability in recombinant P. profundum frr activity across different expression hosts can be attributed to several factors that researchers should systematically investigate:

• Post-translational Modifications: Different host systems provide varying degrees of post-translational modifications necessary for proper protein folding and activity. While E. coli and yeast offer high yields and shorter turnaround times, insect and mammalian cells may provide critical modifications that retain the protein's activity .

• Codon Usage Optimization: P. profundum has a distinctive GC content of approximately 41.3% , which differs from common expression hosts. Analyzing whether codon optimization for the specific expression host improves activity can help determine if translation efficiency affects proper folding.

• Protein Folding Environment: The cytoplasmic environment of different expression hosts varies in terms of pH, ion composition, and chaperone availability. For a marine bacterium like P. profundum, which naturally experiences high salt concentrations, the intracellular environment of the expression host may significantly impact protein folding.

• Purification Method Effects: Different purification strategies may variably affect protein activity. For instance, harsh conditions might be more detrimental to frr expressed in some systems than others.

A systematic comparison of recombinant P. profundum frr expressed in different systems, characterized by both structural (circular dichroism, thermal stability) and functional (ribosome recycling activity) parameters, would help identify the optimal expression system for specific research applications.

How might P. profundum frr interact with ribosome rescue factors under stress conditions?

Investigation of interactions between P. profundum frr and ribosome rescue factors (such as tmRNA and ArfA homologs) under stress conditions represents a promising research direction:

Studies in E. coli have demonstrated that RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA . For P. profundum, which exists in extreme environments and likely experiences various stressors, these interactions may be particularly important for maintaining translational homeostasis.

Future research should:

• Identify Rescue Factor Homologs: Characterize the homologs of tmRNA and ArfA in P. profundum genomes and analyze their expression patterns under different stress conditions (pressure changes, nutrient limitation, temperature shifts).

• Develop Interaction Assays: Establish in vitro and in vivo assays to measure potential physical and functional interactions between frr and rescue factors in P. profundum.

• Stress Response Profiling: Perform ribosome profiling under various stress conditions with and without frr depletion to determine how frr activity modulates the recruitment and action of rescue factors.

The genome sequence data for P. profundum strains provides a foundation for identifying these factors and understanding their potential co-regulation with frr under stress conditions.

What role might P. profundum frr play in adaptation to varying environmental pressures?

The potential role of P. profundum frr in adaptation to varying environmental pressures represents a fascinating area for future research:

Given that P. profundum exists as different ecotypes adapted to varying ocean depths , the frr protein may have evolved specific adaptations to maintain efficient ribosome recycling under different pressure conditions. Research strategies to explore this include:

• Comparative Genomics and Evolution: Analyze frr sequences from multiple P. profundum strains isolated from different depths to identify consistent adaptive mutations associated with pressure tolerance.

• Reciprocal Complementation Experiments: Test whether frr from deep-sea strains can function efficiently at atmospheric pressure and vice versa, using growth rate and protein synthesis rate as functional readouts.

• Structural Biology Approaches: Determine the three-dimensional structures of frr proteins from different P. profundum ecotypes, particularly focusing on pressure-induced conformational changes using high-pressure NMR or crystallography.

• Systems Biology Integration: Investigate whether frr expression or activity is coordinated with other pressure-responsive cellular systems, potentially revealing broader adaptive strategies.

Understanding how essential components of the translation machinery like frr adapt to extreme environmental conditions could provide insights into fundamental mechanisms of protein evolution and function under stress.