Recombinant Frankia sp. Ribosome-recycling factor (frr)

What is Recombinant Frankia sp. Ribosome-recycling factor (frr) and what is its function in protein synthesis?

Recombinant Frankia sp. Ribosome-recycling factor (frr) is a bacterial protein essential for translation termination and ribosome recycling. It functions primarily to dissociate ribosomes from mRNA after the termination of translation, effectively "recycling" ribosomes for subsequent rounds of protein synthesis . The protein, encoded by the frr gene, was originally called ribosome releasing factor but was renamed to ribosome recycling factor to better reflect its function .

In bacterial protein synthesis, RRF works in conjunction with elongation factor G (EF-G) and GTP to disassemble the post-termination complex, releasing the ribosome from the mRNA as either 70S ribosomes or as 50S subunits (leaving 30S subunits on the mRNA) . This recycling process is critical for efficient protein synthesis, as demonstrated by studies showing RRF stimulates in vitro protein synthesis 4- to 7-fold .

How critical is RRF for bacterial survival and growth?

RRF is absolutely essential for bacterial growth and survival. Experimental evidence from E. coli clearly demonstrates this critical role. Researchers constructed an E. coli strain (MC1061-2) carrying a frame-shifted frr gene in the chromosome with a wild-type frr gene on a temperature-sensitive plasmid . This strain exhibited temperature-sensitive growth and could not segregate its frr-carrying plasmid under plasmid incompatibility pressure .

In contrast, control E. coli strains carrying wild-type frr in the chromosome and mutated frr on the temperature-sensitive plasmid showed normal growth at higher temperatures and could segregate their plasmids from incompatible plasmids . Notably, all spontaneously formed thermoresistant colonies derived from the temperature-sensitive strain carried wild-type frr genes that had either been reintegrated into the chromosome or resided on plasmids that had become temperature-resistant . These observations conclusively establish that frr is an essential gene for cell growth.

What expression systems are available for producing Recombinant Frankia sp. RRF in the laboratory?

Researchers have multiple expression system options for producing Recombinant Frankia sp. RRF, each with distinct advantages depending on experimental needs:

The E. coli expression system with Avi-tag biotinylation employs BirA ligase technology to covalently attach biotin to the 15 amino acid AviTag peptide, enabling specific biotinylation in vivo . This system is particularly useful for applications requiring highly specific protein detection or immobilization.

What are the optimal storage conditions for maintaining Recombinant Frankia sp. RRF activity?

To maintain optimal activity of Recombinant Frankia sp. RRF, specific storage conditions must be observed:

• Primary storage should be at -20°C, with extended storage at -20°C or -80°C for maximum stability .

• Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing is not recommended as it may compromise protein activity .

• For reconstitution of lyophilized protein:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended as standard practice)

  • Aliquot for long-term storage at -20°C/-80°C

• Shelf life varies depending on storage conditions:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

These storage recommendations are essential for maintaining protein integrity and functional activity in experimental settings.

How can researchers investigate the role of Recombinant Frankia sp. RRF in translational coupling?

Translational coupling is a process where the translation of one gene influences the translation of an adjacent gene, often involving overlapping termination and initiation codons like UAAUG. To investigate the role of Recombinant Frankia sp. RRF in translational coupling, researchers can employ the following methodologies:

• Reporter Gene Fusion Assays: Create constructs similar to those used in GA phage studies, where the lysis gene translation is coupled to the upstream coat gene translation . By fusing reporter genes like lacZ to the downstream gene and measuring expression levels under conditions of normal and depleted RRF activity, researchers can quantify the impact of RRF on translational coupling.

• Temperature-sensitive RRF Mutant Systems: Develop temperature-sensitive frr mutants in Frankia sp. or use heterologous systems with the Frankia sp. RRF gene. These systems allow for controlled inactivation of RRF function to observe effects on translational coupling without completely eliminating the essential protein .

• In vivo Translation Analysis: Implement ribosome profiling techniques to monitor ribosome positioning on mRNAs containing overlapping termination-initiation regions (like UAAUG) under varying RRF conditions. This approach can reveal how RRF influences ribosome behavior at these junctions .

Studies with model systems have shown that RRF plays a critical role in ensuring correct recognition of the AUG codon as the initiation site for downstream genes in translational coupling scenarios . Similar methodologies can be applied to investigate whether Recombinant Frankia sp. RRF functions analogously.

What techniques can be used to study the interaction between Recombinant Frankia sp. RRF and ribosomal components?

Understanding the molecular interactions between Recombinant Frankia sp. RRF and ribosomal components requires sophisticated biochemical and biophysical approaches:

• Cryo-Electron Microscopy (Cryo-EM): This technique can visualize the interaction of RRF with ribosomal complexes at near-atomic resolution. Previous structural studies have revealed that RRF is a near-perfect mimic of tRNA, providing insights into its functional mechanism .

• Surface Plasmon Resonance (SPR): SPR can measure the binding kinetics and affinity between purified Recombinant Frankia sp. RRF and isolated ribosomes or ribosomal subunits under varying conditions.

• Pull-down Assays with Co-immunoprecipitation: Using antibodies against RRF or ribosomal components, researchers can isolate complexes and identify interacting partners through proteomics approaches.

• Fluorescence Resonance Energy Transfer (FRET): By labeling RRF and ribosomal components with appropriate fluorophores, researchers can monitor dynamic interactions in real-time.

• Biochemical Assays Measuring Ribosome Release: Develop assays that quantify the ability of Recombinant Frankia sp. RRF to release ribosomes from model mRNAs in conjunction with EF-G (or RF3) and GTP . These can be compared with the well-characterized E. coli RRF system to identify functional similarities or differences.

How does RRF function mechanistically in the ribosome recycling process?

The mechanistic function of RRF in ribosome recycling involves a complex interplay with other factors. Based on structural and functional studies, the process occurs as follows:

• Post-termination Complex Formation: After translation termination, the ribosome remains bound to the mRNA with a deacylated tRNA in the P site.

• RRF Binding: RRF, which structurally mimics tRNA, binds to the ribosomal A site .

• EF-G/GTP Interaction: Elongation factor G (EF-G) with GTP then interacts with the RRF-ribosome complex .

• Ribosome Disassembly: The combined action of RRF and EF-G/GTP leads to dissociation of the ribosome from the mRNA, releasing it as either 70S ribosomes or as 50S subunits (leaving 30S subunits on the mRNA) .

• Subsequent Steps: If 30S subunits remain on the mRNA with tRNA, IF3 is necessary to release this tRNA from the 30S subunit .

This process is essential for efficient protein synthesis, as RRF stimulates in vitro protein synthesis 4- to 7-fold . In the absence of RRF, ribosomes not only remain on the mRNA after termination but may also translate portions of the mRNA downstream from the termination codon, potentially leading to aberrant protein synthesis .

What is the potential of Recombinant Frankia sp. RRF as an antimicrobial target?

RRF represents a promising antimicrobial target for several compelling reasons:

• Essentiality: The frr gene is essential for bacterial growth and survival, as definitively demonstrated through genetic experiments . Inhibiting RRF function would therefore be lethal to bacterial cells.

• Structural Uniqueness: While RRF is structurally similar to tRNA, it is a protein target that has no direct counterpart in eukaryotic cells, potentially allowing for selective targeting of bacterial systems.

• Conservation and Specificity: RRF is highly conserved among bacteria but with species-specific variations. These differences could potentially be exploited to develop narrow-spectrum antimicrobials that target specific bacterial pathogens while sparing beneficial microbiota.

• Functional Importance: RRF's critical role in translation termination and ribosome recycling means that even partial inhibition could significantly impair bacterial protein synthesis and growth.

• Existing Precedent: Other translation factors and ribosomal proteins are successfully targeted by existing antibiotics, establishing translation as a validated pathway for antimicrobial intervention.

Research approaches to explore this potential include high-throughput screening of compound libraries against purified Recombinant Frankia sp. RRF, structure-based drug design leveraging the known structure of RRF, and in silico modeling of potential inhibitor binding sites.

How can Recombinant Frankia sp. RRF contribute to our understanding of translational accuracy mechanisms?

RRF plays a role in maintaining translational accuracy during chain elongation , and studying Recombinant Frankia sp. RRF can provide insights into this critical aspect of protein synthesis:

• Comparative Functional Studies: Assess whether Frankia sp. RRF contributes to translational accuracy similar to E. coli RRF by measuring misincorporation rates in in vitro translation systems supplemented with varying amounts of the recombinant protein.

• Structural Analysis: Compare the structure of Frankia sp. RRF with that of other bacterial RRFs to identify conserved domains potentially involved in maintaining translational fidelity.

• Mutational Studies: Generate site-directed mutants of Recombinant Frankia sp. RRF and assess their impact on translational accuracy, helping to map functional domains involved in this process.

• Ribosomal Complex Visualization: Use structural biology techniques to visualize how Frankia sp. RRF interacts with the ribosome during different stages of translation, potentially revealing mechanisms by which it influences accuracy.

Understanding these mechanisms could have implications beyond basic science, potentially informing the development of new approaches to modulate protein synthesis accuracy in biotechnological applications.

How does Frankia sp. RRF compare structurally and functionally to RRF from other organisms?

A comparative analysis reveals both conservation and diversity in RRF structure and function across different organisms:

Every organism examined has a homologue of frr, except for archeons , suggesting that RRF arose after the divergence of bacteria and archaea. The presence of RRF homologs in mitochondria and chloroplasts supports the endosymbiotic theory of their origins from bacterial ancestors.

The conservation of RRF across diverse bacteria while being absent in archaea makes it an interesting subject for studies on the evolution of translation machinery and potentially for the development of broad-spectrum antibacterial compounds.