Recombinant Ribosome-recycling factor (frr)

What is Ribosome-Recycling Factor and what is its primary function in bacteria?

Ribosome-recycling factor (RRF), encoded by the frr gene in Escherichia coli, is responsible for the dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes for subsequent rounds of protein synthesis. RRF works in conjunction with elongation factor G (EF-G) to promote subunit splitting and release of the large ribosomal subunit, enabling ribosomes to participate in new translation cycles .

The mechanism involves two distinct steps: first, the codon-specific release factors (RF1/RF2) recognize stop codons and promote peptidyl-tRNA hydrolysis, releasing the completed polypeptide. Subsequently, RRF acts in concert with EF-G to split the ribosomal subunits, making them available for reinitiation .

How does the molecular mechanism of ribosome recycling differ across the domains of life?

The molecular mechanism of ribosome recycling shows significant evolutionary divergence across the three domains of life:

In bacteria, recycling involves RRF working with EF-G to split the ribosomal subunits after RF1/RF2 have released the nascent peptide. RF3 then removes RF1/RF2, clearing the way for RRF binding. Following subunit splitting, binding of IF3 excludes deacylated tRNA from the 30S subunit and prevents reassembly of the 70S complex .

In eukaryotes and archaea, termination is carried out by a complex containing both a release factor and a translational GTPase (eRF1 and eRF3 in eukaryotes). Despite similar naming, these factors are evolutionarily unrelated to bacterial RFs. After peptide release, eRF1 remains in the ribosome and helps recruit factors that catalyze subunit splitting (Rli1 in yeast or ABCE1 in mammals). The tRNA and small subunit are then released by 40S recycling factors .

This fundamental difference in recycling mechanisms makes bacterial RRF a potential target for antibiotic development, as it represents a process unique to bacteria.

Why is the frr gene considered essential for bacterial cell growth?

Experimental evidence confirms that the frr gene is essential for bacterial viability. Research using a conditional frr expression system in E. coli demonstrated that:

• An E. coli strain (MC1061-2) carrying a frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid exhibits temperature-sensitive growth .

• This strain does not segregate its frr-carrying plasmid under plasmid incompatibility pressure .

• In contrast, isogenic E. coli carrying wild-type frr in the chromosome and mutated frr on a temperature-sensitive plasmid grows normally at elevated temperatures .

• All spontaneously formed thermoresistant colonies derived from the frr-deficient strain carried wild-type frr either through re-exchange in the bacterial chromosome or in a plasmid that became temperature-resistant .

These observations conclusively establish that frr is an essential gene for bacterial cell growth, likely because efficient ribosome recycling is necessary for maintaining adequate translation capacity in growing cells.

What expression systems are most effective for producing recombinant RRF protein?

For recombinant RRF production, E. coli-based expression systems have proven most effective due to several factors:

• Native environment: Since RRF is a bacterial protein, E. coli provides the appropriate cellular machinery for proper folding and post-translational modification.

• Expression conditions: Optimal expression typically involves:

  • BL21(DE3) or similar strains with T7 RNA polymerase

  • IPTG induction at 0.1-0.5 mM when cultures reach OD600 of 0.6-0.8

  • Post-induction growth at 30°C rather than 37°C to enhance solubility

  • Harvest after 3-4 hours of induction

• Purification strategy: A typical workflow includes:

  • Lysis in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, and 5 mM β-mercaptoethanol

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Further purification via ion exchange chromatography

  • Final polishing with size exclusion chromatography

When designing constructs, consideration should be given to the N- and C-terminal regions, as they may affect functionality in biochemical assays. Removal of affinity tags may be necessary for certain structural or functional studies.

How can researchers effectively measure RRF activity in vitro?

Several methodological approaches can be employed to measure RRF activity in vitro:

• Ribosome recycling time measurement:
  Using a complete translation system assembled from purified components (initiation factors, elongation factors, release factors, aminoacyl-tRNA synthetases), researchers can measure the time required for ribosomes to complete successive rounds of translation on synthetic mRNAs. This approach revealed that:

  • Without RF3 and RRF, recycling time is approximately 40 seconds

  • With RF3 alone, recycling time reduces to about 30 seconds

  • With RRF alone, recycling time drops to approximately 15 seconds

  • With both RF3 and RRF, recycling time decreases to less than 6 seconds

• Subunit dissociation assays:

  • Monitoring the conversion of 70S ribosomes to 30S and 50S subunits using sucrose gradient centrifugation

  • Light scattering measurements to track real-time dissociation kinetics

• GTPase activity assays:
  Since RRF works in concert with EF-G, which hydrolyzes GTP, measuring GTP hydrolysis can provide indirect evidence of RRF activity. This can be done using:

  • Radioactive [γ-32P]GTP and thin-layer chromatography

  • Colorimetric assays for phosphate release

When conducting these assays, it's crucial to include appropriate controls and to ensure that the concentrations of all components (ribosomes, RRF, EF-G, GTP) are within physiologically relevant ranges.

What methods can be used to study RRF-ribosome interactions at the molecular level?

Several advanced techniques are available for studying RRF-ribosome interactions at the molecular level:

• Cryo-electron microscopy (cryo-EM):

  • Provides high-resolution structural information of RRF bound to ribosomes

  • Can capture different conformational states during the recycling process

  • Sample preparation requires optimization to trap the desired complexes

• X-ray crystallography:

  • Has been used to determine the structure of isolated RRF

  • Can provide atomic-level details of RRF-ribosome interactions when successful

• Hydroxyl radical footprinting:

  • Identifies regions of rRNA protected by RRF binding

  • Requires expertise in RNA analysis techniques

• Cross-linking mass spectrometry:

  • Uses bifunctional cross-linkers to identify amino acids in close proximity

  • Can map protein-protein interactions between RRF and ribosomal proteins

• Surface plasmon resonance (SPR) and microscale thermophoresis (MST):

  • Determine binding kinetics and affinity constants

  • For SPR, one component (typically ribosomes) is immobilized while RRF flows over the surface

  • MST measures changes in thermophoretic mobility upon binding

• Fluorescence techniques:

  • FRET (Förster Resonance Energy Transfer) to measure distances between labeled components

  • Single-molecule FRET to observe dynamic conformational changes during recycling

When designing these experiments, researchers should consider the physiological relevance of the conditions used, as factors such as buffer composition, temperature, and the presence of other translation factors can significantly influence RRF-ribosome interactions.

How does RRF depletion affect global translation patterns in bacteria?

Ribosome profiling studies have revealed several significant effects of RRF depletion on global translation patterns:

• Ribosome accumulation at termination sites:

  • Upon RRF knockdown, ribosomes accumulate upstream of stop codons

  • Post-termination 70S complexes (post-TCs) fail to be recycled and block elongating ribosomes at the end of open reading frames

• 3'-UTR ribosome density:

  • Dramatic accumulation of ribosome density in 3'-UTRs

  • These appear to be post-TCs that have diffused away from the stop codon over time, not actively translating ribosomes

• Effects on coupled translation:

  • Surprisingly, RRF depletion does not significantly alter the ratio of ribosome density on neighboring genes in polycistronic transcripts

  • This suggests that re-initiation by ribosomes or ribosome subunits bound to mRNA after recycling is not a widespread mechanism of translational initiation in E. coli

• Changes in gene expression:

  • Loss of recycling leads to significant changes in gene expression patterns

  • Notable accumulation of ribosome footprints on transfer-messenger RNA (tmRNA)

  • Dramatic upregulation of ribosome rescue factor ArfA, indicating a cellular response to ribosomal stress

When analyzing ribosome profiling data after RRF depletion, researchers should consider using high-salt conditions (1M NaCl) during lysis to differentiate between elongating ribosomes and post-termination complexes, as high salt dissociates ribosomes into subunits unless they are stabilized by peptidyl-tRNA.

What is the relationship between RRF and other translation factors in the recycling process?

The intricate relationship between RRF and other translation factors reveals a coordinated process:

• Interaction with release factors:

  • RF3 promotes RF1/RF2 cycling between ribosomes, preparing the post-termination complex for RRF binding

  • When both RF3 and RRF are present, ribosome recycling time decreases dramatically (to <6 seconds) compared to either factor alone

• Dependency on EF-G:

  • RRF action is dependent on the concentration of elongation factor-G

  • EF-G provides the GTPase activity necessary for subunit splitting

  • The structural similarity between RRF and tRNA suggests it may mimic tRNA during the recycling process

• Sequential action in termination and recycling:
  The complete process involves several ordered steps:

  • RF1/RF2 recognition of stop codons and peptide release

  • RF3-mediated removal of RF1/RF2

  • RRF binding to the post-termination complex

  • EF-G binding and GTP hydrolysis

  • Ribosome subunit splitting

  • IF3 binding to prevent reassociation of subunits

This ordered sequence suggests that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalyzed consecutively by RF3 and RRF, which are slow in the absence of these factors .

How do structural differences in RRF across bacterial species affect its function?

Structural analyses of RRF from various bacterial species reveal both conserved features and species-specific variations that influence function:

• Domain architecture:

  • All bacterial RRFs consist of two domains: a three-helix bundle (domain I) and a three-stranded β-sheet (domain II)

  • The relative orientation of these domains can vary between species, affecting interactions with ribosomes and EF-G

• Species-specific variations:

  • Sequence identity between bacterial RRFs typically ranges from 40-60%

  • Key residues at the interface with ribosomes and EF-G are generally conserved

  • Surface residues show greater variability, potentially affecting stability and solubility

• Functional implications:

  • Despite structural differences, most bacterial RRFs maintain similar catalytic efficiency

  • Some species-specific RRFs show varying dependencies on EF-G concentration

  • Thermophilic bacteria possess RRFs with enhanced stability at higher temperatures

• Cross-species compatibility:

  • RRF from one species may not function optimally with ribosomes and EF-G from another species

  • This specificity could be exploited for developing species-selective antibiotics targeting RRF

When working with RRF from different bacterial species, researchers should consider these structural differences when designing experiments and interpreting results, especially in cross-species complementation studies.

What is known about RRF homologues in plant chloroplasts and their function?

Plant chloroplasts contain RRF homologues that reflect the endosymbiotic bacterial origin of these organelles:

• Identification and characterization:

  • A protein called mature RRFHCP has been isolated from chloroplasts of spinach (Spinacia oleracea L.)

  • This protein shows 46% sequence identity and 66% sequence homology with bacterial RRF

  • The chloroplastic RRF is encoded by nuclear genes and transported to chloroplasts

• Functional significance:

  • Chloroplastic RRF is essential for chloroplast translation

  • It likely functions similarly to bacterial RRF in recycling chloroplast ribosomes

  • The presence of this factor highlights the conservation of bacterial-type translation machinery in chloroplasts

• Evolutionary implications:

  • The significant sequence homology between chloroplastic and bacterial RRFs supports the endosymbiotic theory

  • The nuclear encoding of chloroplastic RRF demonstrates the transfer of genetic material from endosymbiont to host nucleus during evolution

When studying chloroplastic RRF, researchers should consider the unique environment of the chloroplast and potential adaptations of the recycling mechanism to this organellar context.

How can RRF be used as a target for developing new antimicrobial agents?

The essential nature and bacterial specificity of RRF make it an attractive target for antimicrobial development:

• Target validation:

  • RRF is essential for bacterial growth as demonstrated by conditional lethal mutants

  • The absence of RRF homologues in eukaryotic cytoplasm minimizes potential toxicity issues

  • The bacterial recycling mechanism differs fundamentally from the eukaryotic process

• Screening strategies:

  • In vitro assays measuring RRF activity can be adapted for high-throughput screening

  • Structure-based drug design utilizing crystal structures of RRF alone or bound to ribosomes

  • Fragment-based approaches to identify small molecules that interfere with RRF binding

• Potential advantages as a drug target:

  • Inhibition would lead to ribosome sequestration and translation arrest

  • The high conservation of RRF across bacteria suggests potential broad-spectrum activity

  • Structural differences between RRFs from different bacterial species could be exploited for species-selective agents

• Challenges and considerations:

  • Compounds must penetrate the bacterial cell wall and membrane

  • Potential for resistance development through mutations in RRF or compensatory pathways

  • Need to ensure selectivity against bacterial versus organellar (mitochondrial, chloroplastic) RRFs

When developing RRF-targeting antimicrobials, researchers should focus on disrupting the interaction between RRF and the ribosome or between RRF and EF-G, as these interfaces are critical for function.

What are common issues when expressing recombinant RRF and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant RRF:

• Protein solubility issues:

  • Problem: RRF may form inclusion bodies when overexpressed

  • Solutions:

    • Lower induction temperature (16-25°C)

    • Reduce IPTG concentration (0.1-0.2 mM)

    • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

    • Co-express with molecular chaperones (GroEL/GroES)

• Protein stability concerns:

  • Problem: Purified RRF may show instability during storage

  • Solutions:

    • Include glycerol (10-20%) in storage buffer

    • Add reducing agents like DTT or β-mercaptoethanol

    • Store at higher concentrations (>1 mg/ml)

    • Flash-freeze in liquid nitrogen and store at -80°C in small aliquots

• Activity loss during purification:

  • Problem: RRF may lose activity during purification steps

  • Solutions:

    • Minimize time between purification steps

    • Include stabilizing agents in all buffers

    • Verify activity at each purification stage

    • Consider native purification methods if tag removal affects activity

• Contamination with bacterial ribosomes:

  • Problem: Co-purification of endogenous ribosomes

  • Solutions:

    • Include high salt washes (500 mM NaCl)

    • Add low concentrations of RNase A during lysis

    • Include additional purification steps, such as ion exchange chromatography

    • Validate purity by checking for rRNA contamination via bioanalyzer

When troubleshooting expression issues, a systematic approach testing multiple conditions is recommended, with small-scale expression trials before proceeding to large-scale production.

How can researchers accurately interpret ribosome profiling data in the context of RRF studies?

Interpreting ribosome profiling data in RRF studies requires careful consideration of several factors:

• Distinguishing elongating ribosomes from stalled post-termination complexes:

  • Use high-salt conditions (1M NaCl) during lysis for parallel samples

  • High salt dissociates ribosomes into subunits unless stabilized by peptidyl-tRNA

  • Compare standard and high-salt libraries to identify post-termination complexes

• Normalizing data appropriately:

  • Total library size normalization may not be appropriate if global translation is affected

  • Consider using spike-in controls for normalization

  • Normalize to a subset of genes unlikely to be affected by RRF depletion

• Analyzing footprint distributions:

  • Examine 5' end position of ribosome-protected fragments relative to start and stop codons

  • Look for accumulation patterns at termination sites and in 3'-UTRs

  • Consider codon-specific pausing that may be unrelated to recycling

• Time-course considerations:

  • Collect samples at multiple time points after RRF depletion (e.g., 5, 15, and 60 minutes)

  • Distinguish primary effects from secondary cellular responses

  • Look for progressive changes in ribosome distribution patterns

• Common misinterpretations to avoid:

  • Assuming all 3'-UTR footprints represent active translation

  • Overlooking the effect of ribosome collisions upstream of stalled ribosomes

  • Attributing all changes in translation to direct RRF effects rather than stress responses

When analyzing complex datasets, researchers should consider consulting with computational biologists and statisticians to ensure appropriate data processing and interpretation.

What are the promising research areas for understanding RRF function beyond canonical translation termination?

Several emerging research areas hold promise for expanding our understanding of RRF function:

• Role in ribosome quality control:

  • Investigating RRF involvement in rescuing stalled ribosomes

  • Exploring interactions between RRF and other rescue factors like tmRNA and ArfA

  • Determining if RRF plays a role in ribosome-associated quality control pathways

• Regulatory functions:

  • Examining potential regulatory roles of RRF in stress response

  • Investigating whether RRF activity is modulated under different growth conditions

  • Exploring potential post-translational modifications of RRF that might regulate its function

• Structural dynamics:

  • Using advanced structural biology techniques to capture the complete recycling process

  • Employing single-molecule studies to observe real-time conformational changes

  • Investigating the dynamics of RRF interaction with ribosomes during recycling

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, ribosome profiling)

  • Mathematical modeling of the translation cycle including recycling

  • Network analysis of RRF interactions with the broader translation machinery

• Evolutionary significance:

  • Comparative genomics of RRF across diverse bacterial phyla

  • Investigation of RRF in minimal genomes and symbiotic bacteria

  • Study of RRF adaptations in extreme environments (thermophiles, psychrophiles)

These research directions will benefit from the application of emerging technologies such as cryo-electron tomography, in-cell structural studies, and advanced computational approaches.

How might insights from RRF research contribute to understanding translation in human mitochondria?

Research on bacterial RRF provides valuable insights for understanding mitochondrial translation:

• Mitochondrial recycling mechanisms:

  • Human mitochondria contain an RRF homolog (mtRRF)

  • Investigating similarities and differences between bacterial RRF and mtRRF function

  • Understanding how mtRRF interacts with mitochondria-specific translation factors

• Disease implications:

  • Exploring how mutations in mtRRF contribute to mitochondrial diseases

  • Investigating whether defects in mitochondrial ribosome recycling contribute to aging-related pathologies

  • Developing potential therapeutic strategies for mitochondrial translation defects

• Evolutionary adaptations:

  • Analyzing how mitochondrial RRF has evolved from its bacterial ancestor

  • Understanding adaptations specific to the mitochondrial environment

  • Comparing mitochondrial RRF across different eukaryotic lineages

• Experimental approaches:

  • Developing mitochondria-specific ribosome profiling techniques

  • Creating in vitro reconstituted systems for mitochondrial translation

  • Utilizing patient-derived cells to study mtRRF dysfunction

• Translational medicine applications:

  • Exploring whether bacterial RRF inhibitors might affect mitochondrial translation

  • Investigating potential of mitochondrial RRF as a therapeutic target

  • Developing methods to enhance mitochondrial translation in disease states

This research area represents an important bridge between bacterial translation research and human health applications, with significant potential for understanding fundamental aspects of mitochondrial function and disease.