Recombinant Marinomonas sp. Ribosome-recycling factor (frr)
Description
Biological Role of RRF
RRF collaborates with elongation factor G (EF-G) to split post-termination ribosomes (70S complexes) into subunits (50S and 30S), facilitating ribosome recycling. In Escherichia coli, RRF is essential for viability, as its depletion leads to ribosome stalling at stop codons and disrupted translation efficiency .
Key Functional Features:
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Mechanism: RRF binds to the ribosomal A-site, inducing structural changes that promote subunit dissociation .
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Conservation: Homologs exist across bacteria, including Pseudomonas aeruginosa, where recombinant RRF retains activity in heterologous systems .
Recombinant RRF Production
While no direct studies on Marinomonas sp. RRF are documented, recombinant RRF production in other bacteria involves:
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Gene Cloning: The frr gene is amplified and inserted into expression vectors.
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Host Expression: Commonly expressed in E. coli with affinity tags (e.g., His-tag) for purification .
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Functional Validation: Activity is confirmed via polysome dissociation assays .
Example: Pseudomonas aeruginosa RRF
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Activity: Purified recombinant P. aeruginosa RRF dissociates E. coli polysomes, demonstrating cross-species functionality .
Genomic Context and Regulation
In bacteria, the frr gene is often located in operons with ribosomal proteins (e.g., rpsB, tsf, pyrH) . While Marinomonas sp. genomic data for frr is lacking, CRISPR-Cas systems and bacteriocin synthesis (e.g., R-type bacteriocins) dominate its characterized genetic elements .
Potential Applications and Challenges
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Antibiotic Development: Targeting RRF could disrupt bacterial translation, but its essentiality necessitates caution .
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Biotechnological Tools: Engineered RRF variants might optimize in vitro translation systems.
Challenges:
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Species-Specific Variations: Structural differences in RRF may affect cross-species activity .
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Expression Optimization: Codon usage and solubility require tailoring for Marinomonas sp. .
Research Gaps and Future Directions
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Genomic Characterization: Identification and annotation of frr in Marinomonas sp. genomes.
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Functional Studies: Assays to confirm recombinant RRF activity in native and heterologous systems.
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Structural Analysis: Cryo-EM or X-ray crystallography to resolve Marinomonas RRF-ribosome interactions.
Table 1: Comparative RRF Activity in Bacterial Species
Product Specs
Target Background
KEGG: mmw:Mmwyl1_1275
STRING: 400668.Mmwyl1_1275
Q&A
What is Marinomonas sp. ribosome-recycling factor (frr) and why is it essential for bacterial growth?
Ribosome recycling factor (RRF), encoded by the frr gene, is an essential protein responsible for dissociating ribosomes from mRNA after translation termination. It functions to "recycle" ribosomes, making them available for subsequent rounds of protein synthesis . Studies in Escherichia coli demonstrate that frr is an essential gene for cell growth, as bacterial strains with frame-shifted frr chromosomal copies cannot survive without a functional frr gene provided via a plasmid . In bacterial translation, RRF works together with Elongation Factor G (EF-G) to disassemble the post-termination complex into its constituent ribosomal small (30S) and large (50S) subunits, plus mRNA and free tRNA . This recycling process is crucial for efficient protein synthesis, as evidenced by the dramatic reduction in protein synthesis observed upon loss of RRF both in vivo and in vitro .
How does Marinomonas sp. RRF structure compare to RRF from other bacterial species?
While specific structural data for Marinomonas sp. RRF is not directly available in current literature, comparative analysis can be inferred from characterized bacterial RRFs. Crystal and solution structures of RRF from several organisms show that RRF is composed of two domains that adopt an "L" configuration . The structural conservation of RRF across bacterial species suggests Marinomonas sp. RRF likely maintains this characteristic domain organization.
When bound to ribosomes, RRF induces specific conformational changes in helix H69 in the 50S subunit. Notably, RRF from both E. coli and T. thermophilus causes helix H69 to undergo an ordered to disordered transition when bound to E. coli ribosomes . Additionally, Domain II of RRF directly interacts with protein S12 in the 30S subunit .
What expression systems are most effective for recombinant Marinomonas sp. frr production?
Selecting an appropriate expression system is critical for successful production of functional recombinant Marinomonas sp. RRF. Based on established protocols for similar proteins, the following systems should be considered:
For marine bacterial proteins like those from Marinomonas sp., expression conditions should be optimized to account for potential salt requirements or temperature preferences. Temperature optimization is crucial; many marine proteins show optimal expression at lower temperatures (15-25°C) compared to the standard 37°C used for E. coli.
What purification strategy yields highly active recombinant Marinomonas sp. RRF?
A multi-step chromatographic approach is recommended for purifying recombinant Marinomonas sp. RRF:
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Initial capture using affinity chromatography (if a His-tag or other affinity tag is incorporated)
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Ion exchange chromatography (IEX) to separate based on charge differences
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Size exclusion chromatography (SEC) as a polishing step
For RRF specifically, which interacts with ribosomes, researchers should be cautious about potential co-purification with ribosomal components. A high-salt wash (300-500 mM NaCl) during initial purification steps can help disrupt such interactions. Based on successful purification of other marine-derived recombinant proteins, SEC using a Sephadex G-15 column has proven effective for the final polishing step , and RP-HPLC can be used for final purity assessment.
How can researchers verify the structural integrity of purified recombinant Marinomonas sp. RRF?
Verification of structural integrity is essential to ensure that purified recombinant Marinomonas sp. RRF maintains its native conformation. Multiple complementary techniques should be employed:
For detailed structural analysis, X-ray crystallography or NMR spectroscopy could be employed, similar to structural studies performed on RRF from E. coli and T. thermophilus .
What in vitro assays can accurately measure recombinant Marinomonas sp. RRF activity?
Several complementary assays can be employed to measure the activity of recombinant Marinomonas sp. RRF:
| Assay Type | Methodology | Measured Parameters | Required Components |
|---|---|---|---|
| Ribosome Splitting Assay | Light scattering | Dissociation of 70S ribosomes into subunits | Purified 70S ribosomes, RRF, EF-G, GTP |
| Polysome Disassembly Assay | Sucrose gradient ultracentrifugation | Conversion of polysomes to monosomes and subunits | Isolated polysomes, RRF, EF-G, GTP |
| Translation Termination/Recycling Assay | Scintillation counting or fluorescence | Complete recycling process | Translation components, labeled substrates |
| GTPase Activation Assay | Phosphate release measurement | Stimulation of EF-G GTPase activity | RRF, EF-G, GTP, phosphate detection system |
These assays require purified components, including RRF, EF-G, GTP, and ribosomes. While homologous ribosomes are preferable, cross-species activity is often observed, as noted in the case of T. thermophilus RRF functioning on E. coli ribosomes .
How do environmental factors affect the activity of Marinomonas sp. RRF?
As a marine bacterium, Marinomonas sp. and its proteins, including RRF, have likely evolved adaptations to marine conditions. Environmental factors affecting RRF activity should be systematically investigated:
| Environmental Factor | Expected Optimal Range | Experimental Approach | Significance |
|---|---|---|---|
| Salt Concentration | 0.3-0.5 M NaCl | Activity assays at varying salt concentrations | Adaptation to marine environment |
| Temperature | 15-25°C | Temperature gradient activity profiling | Adaptation to marine temperature ranges |
| pH | 7.5-8.5 (seawater range) | pH gradient activity profiling | Adaptation to marine pH |
| Mg²⁺ Concentration | 5-10 mM | Titration experiments | Critical for ribosome-RRF interaction |
| Pressure | Ambient to 10 MPa | High-pressure chamber experiments | Relevant for depth adaptation |
Since RRF functions in ribosome splitting, structural stability under various conditions can be assessed through monitoring its ability to dissociate 70S ribosomes in conjunction with EF-G. These experiments would reveal adaptations that allow efficient translation in marine environments.
How do mutations in conserved domains affect Marinomonas sp. RRF function?
Mutations in conserved domains would likely significantly impact RRF function. Based on structure-function studies of RRF and related proteins, the following effects can be predicted:
A systematic mutational analysis approach could involve creating point mutations or domain deletions in recombinant Marinomonas sp. RRF, followed by functional assays to assess impact on activity. The approach described for creating the SB1 strain (search result ) provides a model for disrupting conserved domains through homologous recombination.
What is the most effective method for cloning the frr gene from Marinomonas sp.?
Based on successful approaches for cloning genes from marine bacteria, particularly Marinomonas mediterranea , the following strategy is recommended:
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Genomic DNA Extraction: Use a protocol optimized for marine bacteria, potentially including additional salt removal steps.
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PCR Amplification: Design primers based on conserved regions of frr genes from related bacteria. A two-step PCR approach with varying annealing temperatures (10 cycles at 44°C followed by 33 cycles at 48°C) proved successful for amplifying a gene from M. mediterranea .
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Cloning and Sequencing: Clone the PCR product into a vector suitable for both sequencing and expression, such as pGEM-T . Sequence the cloned gene in both directions to ensure accuracy.
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Expression Vector Construction: After sequence verification, subclone the frr gene into an appropriate expression vector, incorporating necessary regulatory elements and purification tags.
This approach leverages both general molecular biology principles and specific successful methods used for cloning genes from Marinomonas species.
How can researchers develop conditional frr mutants in Marinomonas sp.?
Developing frr mutants requires special consideration since frr is likely essential, as demonstrated in E. coli . Based on approaches described in the literature, researchers could:
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Temperature-Sensitive Mutants: Create a strain carrying a frame-shifted chromosomal frr and wild-type frr on a temperature-sensitive plasmid, similar to the E. coli strain MC1061-2 .
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Suicide Vector Approach: Use a suicide vector containing a fragment of the frr gene for insertional mutagenesis through homologous recombination, as described for other genes in M. mediterranea .
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Domain Disruption Strategy: Create partial loss-of-function mutants by disrupting specific conserved domains. This approach involves digesting a plasmid containing the frr gene with restriction enzymes that cut within conserved domains (such as the fifth and sixth conserved domains), creating a deletion, and using this construct for homologous recombination .
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Verification of Essentiality: Test whether thermoresistant colonies derived from conditional mutants carry wild-type frr either in the bacterial chromosome (by re-exchange) or on plasmids that became temperature-resistant, as observed with E. coli frr mutants .
What advanced techniques can be used to study frr regulation in Marinomonas sp.?
Understanding frr regulation requires a combination of molecular and systems biology approaches:
These approaches would provide insights into how frr expression is regulated in response to different environmental conditions or growth phases in Marinomonas sp.
How can recombinant Marinomonas sp. RRF be used to study marine-specific translation adaptations?
Recombinant Marinomonas sp. RRF provides a valuable tool for studying marine-specific adaptations in translation mechanisms:
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Comparative Biochemical Studies: Compare the biochemical properties of Marinomonas sp. RRF with those from non-marine bacteria to identify marine-specific adaptations in stability, activity, and interactions.
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Cross-Species Functionality Tests: Determine whether Marinomonas sp. RRF can function with ribosomes from non-marine bacteria (and vice versa). Such cross-functionality has been observed between T. thermophilus RRF and E. coli ribosomes .
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Structural Studies: Obtain crystal structures of Marinomonas sp. RRF alone and in complex with ribosomes to identify marine-specific structural adaptations, similar to the structural studies of RRF from other bacteria binding to ribosomes .
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Cold Adaptation Analysis: Study how Marinomonas sp. RRF functions at low temperatures compared to RRFs from mesophilic bacteria to reveal adaptations for translation in cold marine environments.
These approaches would contribute to our understanding of how translation processes have adapted to marine environments and could reveal novel mechanisms of protein-ribosome interactions.
What experimental approaches can reveal interactions between Marinomonas sp. RRF and other marine translation factors?
Several experimental approaches can be used to study interactions between Marinomonas sp. RRF and other translation factors:
These approaches would help map the interaction network of Marinomonas sp. RRF and identify any unique interactions specific to marine bacteria that may represent adaptations to marine environments.
How does marine environmental variability affect RRF function in Marinomonas sp.?
Understanding how RRF function in Marinomonas sp. responds to changing marine conditions requires systematic studies under various environmental parameters:
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Temperature Fluctuations: Marine environments experience temperature variations; studying RRF function across a temperature range (4-30°C) would reveal adaptations for thermal flexibility.
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Salinity Changes: Testing RRF activity across a range of salinities would show how coastal Marinomonas species adapt to fluctuating salt concentrations.
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Oxygen Availability: Many marine environments experience oxygen stratification. Examining how RRF function changes under aerobic versus microaerobic conditions would reveal adaptations to such fluctuations.
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Nutrient Limitation: Studying RRF expression and function during nutrient limitation versus replete conditions could reveal regulatory mechanisms that optimize translation efficiency under stress.
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pH Variations: Investigating RRF function across pH ranges relevant to marine environments, including future ocean acidification scenarios, would reveal adaptations or vulnerabilities to pH changes.
These studies would provide insights into how translation recycling in Marinomonas sp. is adapted to function optimally within the variable conditions of marine ecosystems.
