Recombinant Synechococcus sp. Ribosome-recycling factor (frr)

What is the function of ribosome-recycling factor (frr) in Synechococcus sp.?

Ribosome-recycling factor (RRF), encoded by the frr gene, plays a crucial role in bacterial translation by disassembling ribosomes from mRNA after termination of protein synthesis. In cyanobacteria like Synechococcus sp., RRF serves two primary functions:

• Splitting hibernating 100S ribosome complexes: Synechococcus and other bacteria form translationally inactive 70S dimers (100S ribosomes) as a survival strategy during stress conditions. RRF, along with EF-G, synergistically splits these 100S ribosomes in a GTP-dependent manner, allowing them to re-enter the translation cycle .

• Recycling post-termination complexes (PoTc): After translation termination, RRF helps release ribosomes from mRNA and tRNA, making them available for new rounds of protein synthesis .

These functions are essential for bacterial survival, stress responses, and maintaining ribosome integrity. Without effective ribosome recycling, cells experience reduced translational capacity as ribosomes remain unavailable for new protein synthesis .

How does Synechococcus sp. RRF compare structurally and functionally with RRF from other bacteria?

Synechococcus sp. RRF shares fundamental structural and functional characteristics with RRF from other bacterial species, but with notable distinctions:

Structural similarities:

• All bacterial RRFs have a conserved two-domain architecture with a characteristic L-shape

• The N-terminal domain resembles tRNA, allowing RRF to occupy the ribosomal A-site

Functional similarities:

• RRF from Synechococcus sp., like that from E. coli and other bacteria, works in conjunction with EF-G and GTP hydrolysis

• The basic mechanism of ribosome recycling is conserved across bacterial species

Key differences:

• Recent studies suggest RRF from E. coli functions primarily as a ribosome releasing factor rather than a ribosome splitting factor in natural termination

• Cyanobacterial RRFs like that from Synechococcus may have evolved specialized functions related to photosynthetic metabolism and diurnal cycles

• Synechococcus sp. RRF may have unique interactions with cyanobacteria-specific ribosomal proteins and hibernation factors

The role of RRF in cyanobacteria is particularly important in the context of 100S ribosome hibernation, a widespread survival strategy that helps bacteria conserve energy under stress conditions .

What is known about the genomic organization of the frr gene in Synechococcus sp.?

The genomic organization of the frr gene in Synechococcus sp. has several notable characteristics:

• Chromosomal location: In most Synechococcus strains, the frr gene is found on the main chromosome rather than on plasmids.

• Operon structure: The frr gene is often part of a conserved operon that includes genes involved in translation, such as:

  • pyrH (encoding UMP kinase)

  • tsf (encoding elongation factor Ts)

  • frr (encoding ribosome-recycling factor)

• Promoter characteristics: The promoter region of the frr gene in Synechococcus typically contains:

  • A σ70-like promoter sequence

  • Regulatory elements that respond to growth phase and stress conditions

  • Possible light-responsive elements given the photosynthetic nature of cyanobacteria

• Phylogenetic conservation: Comparison of frr genes across Synechococcus strains shows high conservation, reflecting the essential nature of this gene. Different Synechococcus strains have been classified into multiple phylogenetic lineages based on 16S rDNA and phycocyanin operon sequences, with Synechococcus species affiliated to five of eight deeply branching cyanobacterial lineages .

This genomic organization indicates the co-regulation of frr with other translation-related genes, which is logical given their functional relationships in protein synthesis and ribosome recycling.

How do environmental stressors affect RRF expression and function in Synechococcus sp.?

Environmental stressors significantly impact RRF expression and function in Synechococcus sp. through multiple regulatory mechanisms:

• Light/Dark Transitions:

  • RRF gene (frr) expression may be regulated by light/dark cycles, similar to other translation-related genes in cyanobacteria

  • Transcript stability is likely increased during dark periods, as observed with other ribosome-associated proteins like LrtA in Synechocystis sp. PCC 6803, whose transcript half-life is higher in dark-treated cells compared to light-grown cells

• Nutrient Limitation:

  • Under nutrient-limited conditions, Synechococcus increases formation of 100S hibernating ribosomes

  • RRF activity becomes critical during recovery from nutrient limitation, when rapid reactivation of ribosomes is required

  • Phosphate and nitrogen limitation may trigger distinct patterns of RRF regulation

• Temperature Stress:

  • Heat shock induces an alternative ribosome recycling pathway involving HflX GTPase

  • RRF works synergistically with EF-G during normal growth, but under heat stress, the HflX pathway becomes more prominent

  • Cold stress likely increases the formation of 100S ribosomes, making RRF activity essential during recovery

• Methodology for studying stress responses:

  • qRT-PCR to quantify frr transcript levels under different conditions

  • Western blotting with anti-RRF antibodies to monitor protein levels

  • Ribosome profiling to assess ribosome states (70S vs. 100S) under stress

  • Electron microscopy to visualize ribosome dimerization

The intricate regulation of RRF expression and activity ensures that Synechococcus can efficiently manage its translational capacity during environmental fluctuations, conserving energy during stress while maintaining readiness to resume growth when conditions improve .

What are the challenges in expressing functional recombinant Synechococcus sp. RRF in heterologous systems?

Expressing functional recombinant Synechococcus sp. RRF in heterologous systems presents several significant challenges:

• Codon usage bias:

  • Synechococcus sp. has distinct codon preferences compared to common expression hosts like E. coli

  • Methodological solution: Codon optimization of the frr gene sequence for the target expression host is essential, particularly for rare codons

  • Specific approach: Design synthetic gene constructs with host-optimized codons while maintaining the same amino acid sequence

• Post-translational modifications:

  • Potential modifications in native Synechococcus may be absent in heterologous hosts

  • Methodological solution: Compare native and recombinant proteins using mass spectrometry to identify any missing modifications

  • Alternative approach: Consider expression in closely related cyanobacterial hosts

• Protein folding and solubility:

  • The GC-rich DNA of Synechococcus can lead to proteins with different folding requirements

  • Methodological solution: Express with solubility tags (MBP, SUMO, etc.) and optimize induction conditions (temperature, IPTG concentration)

  • Experimental finding: Lower induction temperatures (15-18°C) often improve folding of cyanobacterial proteins

• Functional testing:

  • Confirming activity requires specialized ribosome recycling assays

  • Methodological approach: In vitro ribosome recycling assays using purified components (ribosomes, EF-G, GTP)

  • Control experiment: Compare activity with well-characterized RRF from E. coli

• Expression system optimization:

  • For E. coli expression: The T7/lac or P_trc systems can be used, with the latter showing better performance in some cyanobacterial protein expressions

  • For cyanobacterial expression: Synechococcus sp. PCC 7002 offers a promising homologous expression system with characterized promoter libraries spanning 3 log dynamic ranges

The table below summarizes expression strategies that have been successful for cyanobacterial proteins similar to RRF:

How does Synechococcus RRF interact with hibernating 100S ribosomes versus post-termination complexes?

Synechococcus RRF exhibits distinct interaction mechanisms with hibernating 100S ribosomes compared to post-termination complexes (PoTc):

• Structural differences in binding interfaces:

  • With 100S ribosomes: RRF targets the dimerization interface between two 70S ribosomes, which involves ribosomal proteins and possibly hibernation factors

  • With PoTc: RRF binds to the A-site of a single 70S ribosome containing mRNA and P/E-site tRNA in a rotated conformation

• Biochemical requirements:

  • 100S ribosome splitting: Requires synergistic action of RRF and EF-G in a GTP-dependent but tRNA translocation-independent manner

  • PoTc recycling: Also requires RRF and EF-G with GTP hydrolysis, but the exact order of mRNA and tRNA release remains controversial

• Functional outcomes:

  • 100S splitting outcome: Conversion of inactive dimeric ribosomes into active 70S monomers

  • PoTc recycling outcome: Release of mRNA, tRNA, and dissociation of 70S into 30S and 50S subunits

• Interplay with other factors:

  • 100S ribosomes: Alternative pathway involving heat-shock GTPase HflX exists, especially under stress conditions

  • PoTc: More standardized process that constitutes a fundamental step in the translation cycle

• Kinetic differences:

  • Evidence suggests different binding affinities and processing rates for the two substrates

  • Under normal growth conditions, RRF preferentially targets PoTc, while during recovery from stress, 100S ribosomes become significant targets

This dual functionality of RRF—recycling ribosomes from both PoTc and 100S hibernating complexes—highlights its central role in maintaining translational capacity. The biochemical evidence suggests that while mechanistically similar, these processes have evolved specific recognition elements and regulatory features to respond to different cellular needs .

What are the optimal expression systems and purification methods for recombinant Synechococcus sp. RRF?

The optimal expression systems and purification methods for recombinant Synechococcus sp. RRF involve specific considerations for this cyanobacterial protein:

Expression Systems:

• E. coli-based expression:

  • Recommended strain: BL21(DE3) for standard expression or C43(DE3) for potentially toxic proteins

  • Vector systems: pET series with T7 promoter or pTrc99A with the trc promoter

  • Induction conditions: 0.1-0.5 mM IPTG at 18°C for 16-20 hours to enhance solubility

  • Codon optimization: Essential for high-level expression, especially for rare codons

• Cyanobacterial expression:

  • Synechococcus sp. PCC 7002 offers an excellent homologous expression platform

  • Promoter options:

    • Constitutive: P_cpcB-based promoter library (3 log dynamic range)

    • Inducible: IPTG-inducible system with 48-fold dynamic range outperforms P_trc constructs

  • Integration site: The non-essential acsA locus is suitable for homologous recombination

  • Transformation method: Double homologous recombination with selection using antibiotics (streptomycin/spectinomycin)

Purification Strategy:

• Affinity tags:

  • N-terminal His₆-tag is recommended (C-terminal tags may interfere with function)

  • Alternative tags: MBP fusion for enhanced solubility

• Lysis methods:

  • For E. coli: BugBuster Protein Extraction Reagent has shown efficacy with cyanobacterial proteins

  • For Synechococcus: Sonication in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol

• Chromatography sequence:

  • IMAC (Ni-NTA): Binding buffer with 20-40 mM imidazole to reduce non-specific binding

  • Size exclusion chromatography: To ensure monomeric state and remove aggregates

  • Optional ion-exchange step: For removing nucleic acid contamination

• Tag removal:

  • Recommended protease: TEV protease cleavage site between tag and RRF

  • Post-cleavage purification: Reverse IMAC followed by size exclusion chromatography

• Quality control:

  • SDS-PAGE with Coomassie staining (>95% purity)

  • Mass spectrometry to confirm identity

  • Dynamic light scattering to assess homogeneity

  • Circular dichroism to verify proper folding

This methodological approach combines insights from successful expression of other cyanobacterial proteins with specific considerations for RRF, ensuring high yield and functional quality of the recombinant protein.

How can researchers effectively assess the functional activity of recombinant Synechococcus RRF?

Researchers can effectively assess the functional activity of recombinant Synechococcus RRF using several complementary approaches:

In Vitro Assays:

• Ribosome Splitting Assay:

  • Principle: Measures the ability of RRF to dissociate 70S ribosomes into 30S and 50S subunits

  • Methodology:

    • Incubate purified 70S ribosomes with RRF, EF-G, and GTP

    • Analyze subunit formation by sucrose density gradient centrifugation

    • Quantify 30S and 50S peaks relative to 70S peak

  • Controls: Include reactions without RRF, without EF-G, or without GTP

• 100S Ribosome Disassembly Assay:

  • Principle: Assesses the ability of RRF to convert hibernating 100S ribosomes to active 70S ribosomes

  • Methodology:

    • Prepare 100S ribosomes from stationary phase cultures

    • Incubate with purified RRF, EF-G, and GTP

    • Monitor conversion using sucrose gradient centrifugation or light scattering

  • Quantification: Calculate the rate of 100S to 70S conversion

• GTPase Stimulation Assay:

  • Principle: RRF and EF-G interaction stimulates GTP hydrolysis

  • Methodology:

    • Measure GTP hydrolysis using malachite green assay for phosphate release

    • Compare rates with and without RRF

  • Controls: Include non-hydrolyzable GTP analogs as negative controls

In Vivo Functional Complementation:

• Genetic Complementation:

  • Principle: Test if Synechococcus RRF can rescue an E. coli RRF temperature-sensitive mutant

  • Methodology:

    • Transform E. coli RRF(ts) strain with plasmid expressing Synechococcus RRF

    • Assess growth at non-permissive temperature

  • Controls: E. coli RRF and empty vector as positive and negative controls

• Translational Read-through Assay:

  • Principle: RRF depletion increases stop codon read-through

  • Methodology:

    • Use reporter system with termination codon (as described in search result )

    • Compare GFP fluorescence with and without functional RRF

    • Analyze how Synechococcus RRF affects read-through rates

• Ribosome Profiling:

  • Principle: Measures ribosome distribution on mRNAs

  • Methodology:

    • Compare ribosome footprint patterns in strains with and without functional RRF

    • Analyze accumulation at stop codons as indicator of recycling defects

Data Analysis and Interpretation:

When assessing RRF function, researchers should consider these quantitative parameters:

These methodological approaches provide a comprehensive assessment of RRF functionality, encompassing its roles in both post-termination ribosome recycling and hibernating ribosome reactivation.

What mutagenesis approaches can be used to study structure-function relationships in Synechococcus RRF?

Several mutagenesis approaches can be effectively employed to study structure-function relationships in Synechococcus RRF:

Site-Directed Mutagenesis:

• Methodology:

  • PCR-based mutagenesis using complementary primers containing the desired mutation

  • QuikChange or Q5 Site-Directed Mutagenesis kits are recommended

  • Verification by sequencing both strands of the entire RRF coding region

• Strategic targets:

  • Domain I residues involved in 30S subunit binding

  • Domain II residues important for 50S interaction

  • Hinge region residues affecting interdomain flexibility

  • Residues at the putative EF-G interaction interface

• Functional assessment:

  • Compare activity of wild-type and mutant proteins in ribosome splitting assays

  • Measure binding affinities to 70S and 100S ribosomes

  • Assess interaction with EF-G using pull-down assays

Random Mutagenesis:

• Methodology:

  • Error-prone PCR using GeneMorph II Random Mutagenesis Kit as described for promoter libraries

  • Multiple rounds of error-prone PCR using low template amounts (0.1 ng) to maximize mutation frequency

  • Creation of a mutant library followed by functional screening

• Library screening approaches:

  • Complementation of temperature-sensitive RRF mutant in E. coli

  • High-throughput ribosome recycling assays using fluorescence-based reporters

  • Selective pressure in Synechococcus under stress conditions

• Analysis:

  • Sequencing of functional and non-functional variants

  • Identification of mutation hotspots critical for function

  • Statistical analysis of mutation patterns

Domain Swapping:

• Methodology:

  • Design chimeric proteins combining domains from Synechococcus RRF and other bacterial RRFs

  • Use overlap extension PCR to generate seamless domain fusions

  • Express and purify chimeric proteins using the same protocols as wild-type

• Strategic swaps:

  • Domain I from E. coli RRF with Domain II from Synechococcus RRF

  • Hinge region swaps to investigate flexibility requirements

  • Surface loop replacements to study species-specific interactions

• Functional analysis:

  • Compare activity in homologous versus heterologous systems

  • Assess the role of each domain in ribosome binding and recycling

Truncation Analysis:

• Methodology:

  • Generate systematic N-terminal and C-terminal truncations

  • Create internal deletions of specific structural elements

  • Express as His-tagged constructs for parallel purification

• Key constructs:

  • Domain I alone (tRNA-mimicry domain)

  • Domain II alone (ribosome-binding domain)

  • Variants with altered interdomain linker length

• Structural assessment:

  • Circular dichroism to verify folding

  • Limited proteolysis to identify stable domains

  • Size exclusion chromatography to assess oligomeric state

Cysteine Scanning Mutagenesis:

• Methodology:

  • Replace individual residues with cysteine in a cysteine-free RRF background

  • Label with environment-sensitive fluorophores

  • Monitor conformational changes during function

• Applications:

  • Mapping conformational changes during ribosome interaction

  • Probing the dynamic range of interdomain movement

  • FRET studies to measure distances between labeled positions

These mutagenesis approaches, when combined with detailed functional assays, provide powerful tools for dissecting the structure-function relationships in Synechococcus RRF, particularly its roles in hibernating ribosome reactivation and post-termination ribosome recycling.

How does the activity of Synechococcus RRF compare across different strains and evolutionary lineages?

The activity of Synechococcus RRF shows significant variation across different strains and evolutionary lineages, reflecting adaptations to diverse ecological niches:

Phylogenetic Distribution:

Synechococcus species are distributed across five of eight deeply branching cyanobacterial lineages, as revealed by 16S rDNA sequence analysis . This phylogenetic diversity is mirrored in RRF structural and functional variations:

• Marine vs. Freshwater Strains:

  • Marine Synechococcus strains (e.g., WH8102, WH7803) have RRFs adapted to higher salt concentrations

  • Freshwater strains (e.g., PCC 7942) possess RRFs with different surface charge distributions

  • Functional consequence: Marine RRFs typically maintain activity at higher ionic strengths

• Thermophilic vs. Mesophilic Strains:

  • Hot-spring isolates (including PCC 6716 and PCC 6717) form a distinct cyanobacterial lineage

  • Their RRFs contain adaptations for thermostability, including:

    • Higher proportion of charged residues forming salt bridges

    • Increased hydrophobic core packing

    • Reduced flexibility in certain loop regions

  • Functional consequence: Thermophilic RRFs maintain activity at elevated temperatures but often show reduced flexibility

Methodological Approaches for Comparative Analysis:

• Sequence-based comparison:

  • Multiple sequence alignment of RRF proteins from diverse Synechococcus strains

  • Identification of conserved residues (core function) versus variable regions (adaptation)

  • Calculation of selection pressures (dN/dS ratios) to identify sites under positive selection

• Biochemical comparison:

  • Side-by-side activity assays under standardized conditions

  • Temperature and pH activity profiles for RRFs from different lineages

  • Cross-species complementation experiments

• Structural biology approaches:

  • Comparative homology modeling based on available bacterial RRF structures

  • Analysis of surface electrostatics across evolutionary lineages

  • Molecular dynamics simulations to compare flexibility and conformational repertoire

Evolutionary Insights:

The comparative analysis of Synechococcus RRFs reveals that while core catalytic functions are conserved, significant adaptations have occurred to accommodate:

• Different ribosomal RNA and protein compositions across lineages

• Varying environmental conditions (temperature, salinity, pH)

• Diverse metabolic strategies (obligate photoautotrophy vs. facultative heterotrophy)

• Specialized stress response mechanisms

This evolutionary plasticity in RRF function may contribute to the ecological success of Synechococcus across diverse aquatic environments while maintaining the essential role in ribosome recycling.

What experimental approaches can be used to study the role of RRF in cyanobacterial stress responses?

Multiple experimental approaches can be employed to elucidate the role of RRF in cyanobacterial stress responses, with each providing unique insights:

Genetic Manipulation Approaches:

• Controlled RRF Depletion System:

  • Methodology: Replace native frr promoter with an inducible promoter (such as P_trc-derived systems with 48-fold dynamic range )

  • Analysis: Monitor physiological effects during gradual RRF depletion under various stresses

  • Expected outcomes: RRF depletion reduces translation capacity and prevents new rounds of translation

• Overexpression Studies:

  • Methodology: Express RRF under strong promoters (e.g., P_cpcB from Synechococcus PCC 7002)

  • Integration: Target non-essential genomic loci like nrsBACD operon using homologous recombination

  • Analysis: Assess impact on ribosome hibernation, stress tolerance, and recovery

• Site-Directed Mutagenesis:

  • Target: Create variants with altered affinity for 100S ribosomes versus PoTc

  • Approach: Introduce mutations at the EF-G interaction interface

  • Analysis: Compare stress survival between wild-type and mutant strains

Biochemical and Structural Approaches:

• In Vitro Reconstitution:

  • Methodology: Purify components (ribosomes, RRF, EF-G) and reconstitute recycling under stress-mimicking conditions

  • Variables: Test effects of temperature, pH, salt concentration, and molecular crowding

  • Analysis: Quantify recycling rates and efficiency using sucrose gradient analysis

• Cryo-Electron Microscopy:

  • Target: Structure of Synechococcus 100S ribosomes with and without RRF/EF-G

  • Resolution: Near-atomic resolution to visualize molecular interactions

  • Outcome: Mechanistic insights into how RRF disrupts 100S dimer interface

• Protein-Protein Interaction Studies:

  • Techniques: Bacterial two-hybrid, pull-down assays, surface plasmon resonance

  • Targets: Interaction between RRF and stress-specific factors

  • Analysis: Identify stress-specific interaction partners that may regulate RRF activity

Systems Biology Approaches:

• Ribosome Profiling:

  • Methodology: Deep sequencing of ribosome-protected mRNA fragments

  • Comparison: Wild-type versus RRF-depleted cells under various stresses

  • Analysis: Identify genes differentially translated during stress and recovery

• Proteomics:

  • Approach: Quantitative mass spectrometry of wild-type versus RRF-mutant strains

  • Focus: Global protein synthesis patterns during stress and recovery phases

  • Analysis: Identify protein subsets most affected by RRF dysfunction

• Transcriptomics:

  • Method: RNA-seq of frr mutants under various stress conditions

  • Analysis: Assess transcriptional responses that compensate for altered RRF activity

  • Integration: Combine with ribosome profiling data to identify translationally regulated genes

Stress-Specific Experimental Designs:

These multifaceted approaches provide complementary insights into how RRF contributes to stress survival in Synechococcus and other cyanobacteria by modulating ribosome activity to conserve energy while maintaining translational readiness for stress recovery.

How can engineered variants of Synechococcus RRF be used to optimize protein expression in cyanobacterial biotechnology?

Engineered variants of Synechococcus RRF can significantly enhance protein expression in cyanobacterial biotechnology through strategic modifications that optimize ribosome recycling and translational efficiency:

RRF Engineering Strategies:

• Affinity-Optimized Variants:

  • Approach: Create RRF variants with enhanced binding to post-termination complexes

  • Methodology: Structure-guided mutations at the ribosome-binding interface

  • Expected outcome: Faster ribosome turnover and increased translation rates

• Stress-Decoupled Variants:

  • Approach: Engineer RRF to resist inactivation during stress conditions

  • Methodology: Identify and modify regulatory sites that modulate RRF activity under stress

  • Benefit: Maintain protein expression during suboptimal culture conditions

• Hibernation-Resistant Variants:

  • Approach: Create RRF variants that preferentially disassemble 100S ribosomes

  • Mechanism: Enhanced interaction with the 100S dimer interface

  • Application: Prevent ribosome hibernation during stationary phase to extend production time

Expression System Optimization:

• Co-expression Strategies:

  • Components: Optimized RRF + EF-G co-expression from a single construct

  • Promoter design: Use characterized strong promoters like P_cpcB with 3-log dynamic range

  • Vector design: Integrate into genomic loci like acsA using homologous recombination

• Synthetic Regulatory Circuits:

  • Design: Create tunable RRF expression systems using characterized genetic parts for Synechococcus

  • Components: Combine synthetic constitutive promoter libraries with engineered RBS sequences

  • Control: IPTG-inducible systems with 48-fold dynamic range outperform P_trc constructs

• Translational Enhancement:

  • Approach: Co-optimize RRF and target protein expression using synthetic biology tools

  • Elements: Designer RBS sequences optimized for Synechococcus

  • Structure: Implement translation coupling systems for coordinated expression

Implementation Strategies:

• Genetic integration approach:

  • Target non-essential genetic loci like nrsBACD for stable integration

  • Use streptomycin/spectinomycin resistance for selection

  • Confirm integration by PCR and Southern blot analysis

• Experimental design for optimization:

  • Factorial experiments varying RRF variant type and expression level

  • Reporter systems using fluorescent proteins like YFP

  • Standardized measurements normalizing to OD₇₃₀

• Productivity assessment:

  • Monitor target protein yield using quantitative Western blotting

  • Assess culture viability and longevity during production

  • Measure translation efficiency using polysome profiling

Case-Specific Applications:

By rationally engineering RRF and integrating it into optimized expression systems, researchers can overcome translational bottlenecks in cyanobacterial biotechnology, potentially transforming these photosynthetic organisms into more efficient bioproduction platforms.

What insights from Synechococcus RRF studies can inform our understanding of antibiotic resistance mechanisms?

Studies on Synechococcus RRF provide valuable insights into antibiotic resistance mechanisms, particularly those involving the translational machinery:

RRF as an Antibiotic Target:

• RRF is essential for bacterial survival and absent in eukaryotes, making it an attractive antibiotic target

• Structural studies of Synechococcus RRF reveal:

  • Unique binding pockets that could be exploited for cyanobacteria-specific inhibitors

  • Conserved functional regions shared with pathogenic bacteria

  • Structural differences from human mitochondrial RRF that could enable selectivity

• Drug development implications:

  • RRF inhibitors would target a different step in translation than existing antibiotics

  • Potential for synergistic effects when combined with other translation inhibitors

  • Reduced likelihood of existing resistance mechanisms affecting RRF-targeting drugs

Ribosome Hibernation and Antibiotic Tolerance:

• Hibernating 100S ribosomes in Synechococcus show altered antibiotic susceptibility patterns:

  • 100S ribosomes are less accessible to certain ribosome-targeting antibiotics

  • RRF's role in disassembling 100S ribosomes affects antibiotic efficacy

• Persister cell formation:

  • Ribosome hibernation is linked to bacterial persistence

  • RRF's involvement in reversing hibernation is intimately linked to bacterial pathogenesis and persister formation

  • Understanding this process in Synechococcus provides a model for similar mechanisms in pathogens

• Methodological approaches:

  • Monitor antibiotic efficacy in strains with altered RRF expression

  • Compare minimum inhibitory concentrations during active growth versus stress conditions

  • Track ribosome states (70S vs. 100S) during antibiotic exposure

RRF Mutations and Resistance Mechanisms:

• Natural variation in RRF across Synechococcus strains reveals:

  • Regions under selective pressure that may relate to antibiotic resistance

  • Adaptations that maintain function despite structural changes

  • Potential cross-resistance patterns between different classes of translation inhibitors

• Resistance-conferring mutations:

  • Mutations affecting the RRF-ribosome interaction may alter antibiotic binding sites

  • Changes in RRF-EF-G interaction could affect susceptibility to EF-G inhibitors

  • Synechococcus can serve as a non-pathogenic model for studying these mutations

• Experimental approaches:

  • Generate and characterize RRF mutations that confer resistance to translation inhibitors

  • Perform structural studies to understand how mutations affect inhibitor binding

  • Use directed evolution to identify potential resistance pathways

Translation Quality Control and Antibiotic Effects:

• RRF's role in preventing translational read-through has implications for:

  • Antibiotics that induce miscoding or read-through

  • Quality control mechanisms that detect aberrant translation products

  • Stress responses triggered by translation errors

• Methodological insights:

  • Reporter systems for measuring stop codon read-through in the presence of antibiotics

  • Approaches to quantify translational fidelity during stress and recovery

  • Techniques to monitor ribosome recycling dynamics in vivo

These insights from Synechococcus RRF studies provide valuable contributions to our understanding of translation-targeting antibiotics and bacterial resistance mechanisms, potentially informing the development of novel antimicrobial strategies that could address the growing challenge of antibiotic resistance.