Recombinant Salmonella paratyphi B Ribosome-recycling factor (frr)

What is Ribosome-recycling factor (frr) in Salmonella paratyphi B?

Ribosome-recycling factor (RRF), encoded by the frr gene in Salmonella paratyphi B, is an essential protein that functions in the final stage of protein synthesis. RRF is responsible for releasing ribosomes from mRNA after termination of translation, allowing them to be recycled for subsequent rounds of protein synthesis. Similar to RRF in other bacterial species, Salmonella paratyphi B RRF likely consists of approximately 185 amino acids arranged in an L-shaped structure that remarkably resembles tRNA . This structural mimicry is fundamental to its function in recycling post-termination ribosomal complexes.

Research has conclusively demonstrated that RRF is essential for bacterial survival, as bacteria cannot survive without functional RRF . The protein works in conjunction with elongation factor G (EF-G) and requires GTP hydrolysis to facilitate ribosome recycling.

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

The structure of RRF exhibits a remarkable near-perfect mimicry of tRNA in both shape and size, which is critical to its function. Crystallographic studies have revealed that RRF proteins display a characteristic L-shaped configuration that closely matches the dimensions of tRNA . This structure consists of two distinct domains:

• Domain I: A three-helix bundle that forms the vertical arm

• Domain II: A three-stranded beta-sheet that forms the horizontal arm

This structural arrangement enables RRF to bind to the ribosomal A-site after peptide release during termination. The binding position of RRF relative to tRNA in the 70S ribosome is crucial for its mechanism of action . While RRF structurally mimics tRNA, it lacks an anticodon arm, which aligns with its function of binding to ribosomes independently of mRNA codons.

The structural mimicry allows RRF to interact with the ribosome in a position similar to tRNA, which is essential for its role in dissociating post-termination ribosomal complexes in concert with EF-G and GTP hydrolysis.

What is the role of RRF in protein synthesis termination?

RRF plays a critical role in the terminal phase of protein synthesis, specifically in recycling ribosomes after the termination event. The process occurs in several sequential steps:

• During termination, release factors RF1 or RF2 recognize stop codons (UAA, UAG, or UGA) on the mRNA and catalyze the hydrolysis of the peptidyl-tRNA bond, releasing the completed polypeptide chain.

• This leaves behind a post-termination complex consisting of the ribosome still bound to mRNA with a deacylated tRNA in the P-site.

• RRF binds to the ribosomal A-site, mimicking tRNA.

• EF-G then binds and hydrolyzes GTP, providing energy for the conformational changes.

• These changes lead to the release of mRNA and deacylated tRNA from the ribosome.

• The ribosome subsequently dissociates into its 30S and 50S subunits, which are then available for new rounds of translation .

Recent research indicates that RRF functions primarily as a "ribosome releasing factor" rather than merely a "ribosome splitting factor," suggesting its main function is to release ribosomes from mRNA at the termination codon .

Efficiency studies in E. coli have shown that the presence of both RRF and RF3 (another release factor) dramatically reduces the "recycling time" of ribosomes from approximately 40 seconds (in the absence of both factors) to less than 6 seconds . This demonstrates the significant efficiency enhancement these factors provide to the recycling process.

How is RRF involved in translational coupling in bacterial systems?

Translational coupling refers to the phenomenon where the translation of a downstream open reading frame (d-ORF) depends on the translation of an upstream open reading frame (u-ORF). This occurs frequently in bacteria, particularly in operons where genes have overlapping stop and start codons (e.g., UAAUG sequences).

Research has demonstrated that RRF plays a significant role in this process by releasing ribosomes from mRNA at the termination codon of the u-ORF. Experimental evidence shows that in the absence of RRF, more ribosomes that finished translating the u-ORF were used for reading the d-ORF, indicating that RRF normally functions to release these ribosomes from the mRNA .

Interestingly, studies have revealed that even in the presence of RRF, ribosomes do not always completely leave the mRNA after termination but may translate the same mRNA multiple times. This suggests that RRF does not actively eject mRNA from terminating ribosomes but rather makes them mobile on the mRNA and ready to enter the next translation round .

An additional finding is that short u-ORFs (less than 5 codons) inhibited d-ORF reading by ribosomes finishing u-ORF translation, suggesting that the termination process in short ORFs differs from that in normal-length ORFs . This has important implications for understanding gene expression regulation in operons and other closely spaced genes in bacterial genomes, including Salmonella paratyphi B.

What methodological approaches are optimal for expressing and purifying recombinant Salmonella paratyphi B RRF?

Based on established protocols for similar proteins, the following methodological approach is recommended for expressing and purifying recombinant Salmonella paratyphi B RRF:

Table 1: Expression and Purification Protocol for Recombinant Salmonella paratyphi B RRF

For reconstitution of lyophilized protein, it is recommended to reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For working with the protein, aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided .

How does RRF interact with elongation factor G (EF-G) to facilitate ribosome recycling?

The interaction between RRF and EF-G is critical for efficient ribosome recycling. This process involves a coordinated sequence of molecular events:

Molecular Mechanism:

• RRF binds to the A-site of the post-termination ribosome complex, adopting a position that mimics tRNA.

• EF-G then binds to the same ribosome, creating a transient RRF-EF-G-ribosome complex.

• The interaction between RRF and EF-G triggers conformational changes in both the factors and the ribosome.

• These conformational changes, powered by GTP hydrolysis, lead to the dissociation of the ribosomal subunits .

Role of GTP Hydrolysis:
GTP hydrolysis by EF-G is essential for this process. Experimental evidence using non-hydrolyzable GTP analogues has shown that mRNA is not released when GTP cannot be hydrolyzed, preventing successful ribosome recycling . This demonstrates that the energy from GTP hydrolysis drives the conformational changes needed for ribosome dissociation.

Kinetics of the Process:
In E. coli, the presence of both RRF and RF3 (another release factor) dramatically reduces the ribosome recycling time from approximately 40 seconds (in the absence of both factors) to less than 6 seconds . This indicates that these factors work synergistically to make the recycling process much more efficient.

Concentration Dependence:
The action of RRF depends on the concentration of EF-G, highlighting the cooperative nature of their interaction . This suggests that the stoichiometry of RRF to EF-G is important for efficient ribosome recycling.

Structural Basis of Interaction:
While specific structural details for Salmonella paratyphi B proteins are not fully characterized, studies in related bacteria indicate that domain II of RRF interacts with domain IV of EF-G. These interactions are likely conserved in Salmonella paratyphi B given the high conservation of these proteins across bacterial species.

Understanding these interactions is not only academically valuable but also has potential applications in antimicrobial development, as the RRF-EF-G interaction represents a unique target that differs from those of existing antibiotics.

What experimental models are most effective for studying RRF function in Salmonella paratyphi B?

Several experimental models have proven valuable for studying RRF function in bacterial systems and can be applied to Salmonella paratyphi B research:

In Vitro Translation Systems:
Reconstituted translation systems assembled from purified components (initiation factors, elongation factors, termination factors, and aminoacyl-tRNA synthetases) allow for controlled studies of RRF function . These systems enable:

• Measurement of ribosome recycling time

• Analysis of the effects of RRF mutations

• Study of interactions between RRF and other translation factors

• Quantitative assessment of the impact of potential RRF inhibitors

Translational Coupling Models:
Both in vivo and in vitro translational coupling systems provide insights into RRF function:

• In vivo systems using reporter genes with shared termination-initiation sequences (e.g., UAAUG)

• In vitro translational coupling with synthetic mRNAs containing junction sequences

• Reporter systems using lacZ fusions to measure downstream gene expression

These models are particularly useful for understanding RRF's role in the transition between termination and initiation of translation in adjacent genes.

Genetic Approaches:

• Temperature-sensitive RRF mutants allow controlled inactivation of RRF function in vivo

• Conditional knockdown of frr expression using regulatable promoters

• Site-directed mutagenesis to study specific residues

• Complementation studies with mutant frr genes

Structural and Biochemical Studies:

• X-ray crystallography or cryo-electron microscopy to determine structures

• Surface plasmon resonance (SPR) to measure binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic analysis

• Cross-linking studies to identify interaction sites

Host-Pathogen Interaction Models:
For studying RRF in the context of Salmonella pathogenesis:

• Cell culture infection models (epithelial cells, macrophages)

• Mouse infection models

• Analysis of bacterial fitness in competitive infection assays

Comparative Genomics:
Analysis of frr gene sequences across Salmonella serovars can identify conserved and variable regions that might correlate with pathogenicity or host adaptation .

How could mutations in the frr gene affect pathogenicity of Salmonella paratyphi B?

Mutations in the frr gene could influence Salmonella paratyphi B pathogenicity through several mechanisms:

Translational Efficiency Impact:
Since RRF is essential for efficient ribosome recycling, mutations affecting its function could impair translation efficiency, resulting in:

• Reduced growth rates during infection

• Altered synthesis of virulence factors

• Compromised competitive fitness in the host environment

Stress Response Alterations:
RRF activity may be particularly important under stress conditions encountered during infection:

• Nutrient limitation environments

• Temperature fluctuations due to host fever response

• Oxidative stress from host immune defenses

Mutations affecting RRF function under these conditions could impair the pathogen's ability to adapt and persist in the host.

Virulence Gene Expression:
If virulence genes in Salmonella paratyphi B are organized in operons with translational coupling, mutations in frr could affect the expression ratios of these genes . This could alter the balance of virulence factors produced during infection, potentially changing the disease presentation or severity.

Persistence Capabilities:
Some Salmonella species can enter a viable but non-culturable (VNC) state under adverse conditions . If RRF plays a role in recovery from this state, mutations could affect the ability of Salmonella paratyphi B to persist in the environment or host and later reactivate to cause disease.

Antibiotic Susceptibility:
Since RRF is involved in protein synthesis, mutations could potentially affect susceptibility to antibiotics targeting the translation machinery, including aminoglycosides, macrolides, or tetracyclines. This could influence treatment outcomes and the development of antibiotic resistance.

Understanding these relationships could aid in the development of attenuated vaccine strains, as mutations in frr might provide a balance between attenuation and immunogenicity, potentially contributing to vaccine development efforts such as those described for Salmonella Paratyphi B .

What are the implications of RRF inhibition for antimicrobial development against Salmonella paratyphi B?

RRF represents a promising target for novel antimicrobial development against Salmonella paratyphi B for several compelling reasons:

Essential Target for Viability:
Bacteria do not survive without functional RRF, making it an attractive target for antimicrobial development . Inhibition of RRF would be lethal to the bacteria rather than merely bacteriostatic.

Selective Toxicity Potential:
RRF is highly conserved across bacterial species but has no direct homolog in eukaryotic cells. This offers potential for selective toxicity:

• Broad-spectrum antibacterial activity

• Minimal toxicity to human cells

• Reduced impact on non-targeted physiological processes

Unique Molecular Target:
The distinctive structure of RRF with its tRNA mimicry provides a target that differs from existing antibiotic targets . This uniqueness could help overcome resistance to current antibiotics by exploiting a completely different mechanism.

Potential Inhibition Strategies:

Combination Therapy Potential:
Inhibitors of RRF could potentially synergize with existing antibiotics, particularly those that target other aspects of protein synthesis. This approach could:

• Allow for lower doses of existing antibiotics

• Reduce side effects

• Potentially overcome some forms of resistance

• Provide more effective clearance of infection

Development Challenges:
Several obstacles must be addressed:

• Achieving sufficient cellular penetration through the double membrane of Gram-negative bacteria

• Avoiding efflux by bacterial pumps

• Developing compounds with suitable pharmacokinetic properties

• Identifying inhibitors with adequate selectivity

Advances in the development of RRF inhibitors could provide valuable new therapeutic options for treating Salmonella paratyphi B infections, particularly in the context of increasing antimicrobial resistance.