Recombinant Rhodobacter sphaeroides Elongation factor G (fusA), partial
Description
Overview of Elongation Factor G (EF-G)
EF-G is a GTPase essential for ribosomal translocation during protein synthesis. It facilitates the movement of tRNA and mRNA through the ribosome and participates in ribosome recycling. Key features include:
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Domain Structure: Five domains (I–V), with domain I binding GTP and domains III–V interacting with the ribosome .
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Functional Role: Drives tRNA translocation post-peptide bond formation and splits ribosomes into subunits during recycling .
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Antibiotic Target: Fusidic acid (FA) inhibits EF-G by trapping it in a GDP-bound state on the ribosome, blocking turnover .
Recombinant EF-G in Rhodobacter sphaeroides
While R. sphaeroides EF-G has not been explicitly characterized in recombinant form, its homologs in other bacteria (e.g., Staphylococcus aureus, Synechocystis) provide mechanistic insights:
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FA Resistance: Mutations in fusA (e.g., F88L in S. aureus) reduce FA binding by altering EF-G conformation, compromising antibiotic efficacy but often incurring fitness costs .
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Redox Regulation: In Synechocystis, EF-G activity is modulated by cysteine oxidation states, with thioredoxin restoring function under oxidative stress . This redox sensitivity may extend to R. sphaeroides given its photosynthetic lifestyle .
Comparative Functional Data
Data from EF-G homologs highlight critical residues and dynamics:
Research Gaps and Future Directions
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Structural Data: No high-resolution structures of R. sphaeroides EF-G are available. Cryo-EM studies of its photosynthetic machinery (e.g., LH1-RC complexes ) suggest methodologies applicable to EF-G.
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Redox Sensitivity: R. sphaeroides EF-G may integrate with thioredoxin systems under light stress, akin to Synechocystis .
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Engineering Applications: Partial EF-G variants could optimize translational efficiency or antibiotic resistance in synthetic biology workflows.
Key References
Product Specs
Target Background
KEGG: rsk:RSKD131_0011
Q&A
Basic Research Questions
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What is Elongation factor G (fusA) and what is its function in bacteria?
Elongation factor G (EF-G), encoded by the fusA gene, is a critical protein involved in bacterial protein synthesis. It primarily functions during the elongation phase of translation by catalyzing the translocation step, where it moves the peptidyl-tRNA from the A-site to the P-site of the ribosome. Additionally, EF-G works together with ribosome recycling factor (RRF) to dissociate ribosomes from mRNA after translation termination, enabling the ribosomal subunits to participate in new rounds of protein synthesis . The protein plays a crucial role in maintaining efficient translation rates and ensuring proper protein synthesis in bacteria.
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What is the molecular weight and structure of R. sphaeroides Elongation factor G?
The recombinant partial Elongation factor G (fusA) from Rhodobacter sphaeroides has a molecular weight of approximately 77,720 Da . The protein belongs to the GTPase superfamily and typically contains five domains (I-V). Domain I exhibits GTPase activity and shares structural similarities with other GTPases, while domains II-V are involved in interactions with the ribosome during translocation. The protein undergoes conformational changes during GTP hydrolysis that are essential for its function in ribosomal translocation.
Domain Function Approximate Size I GTPase activity, GTP binding ~200 amino acids II Ribosome interaction ~100 amino acids III Ribosome interaction ~100 amino acids IV mRNA/tRNA interaction ~110 amino acids V Ribosome interaction ~90 amino acids -
What expression systems are commonly used for producing recombinant fusA?
The most common expression system for producing recombinant Rhodobacter sphaeroides Elongation factor G (fusA) is Escherichia coli . This bacterial expression system offers several advantages, including rapid growth, high protein yields, and well-established protocols for induction and purification. When expressing fusA in E. coli, researchers typically use vectors with inducible promoters such as T7 or tac to control expression. The protein may be expressed with various affinity tags to facilitate purification. Selection of an appropriate E. coli strain is crucial, with BL21(DE3) and its derivatives being commonly used due to their reduced protease activity and ability to express T7 RNA polymerase.
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How should recombinant fusA protein be stored and handled for optimal stability?
For optimal stability of recombinant Rhodobacter sphaeroides Elongation factor G (fusA), proper storage conditions are essential. The lyophilized form of the protein has a shelf life of approximately 12 months when stored at -20°C to -80°C, while the liquid form typically maintains stability for about 6 months under the same conditions . For working aliquots, storage at 4°C is recommended for up to one week to avoid repeated freeze-thaw cycles, which can compromise protein integrity .
When reconstituting lyophilized protein, it should be briefly centrifuged prior to opening, and then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% is recommended before aliquoting for long-term storage at -20°C or -80°C . The protein is sensitive to environmental factors such as heat, light, and certain chemicals, so appropriate handling practices should be followed to maintain its structural and functional integrity.
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What is the difference between partial and full-length fusA protein?
The partial Rhodobacter sphaeroides Elongation factor G (fusA) protein represents a fragment of the complete protein, containing only specific domains or segments rather than the entire amino acid sequence . This partial version may be designed to include specific functional domains of interest while excluding others, which can be advantageous for certain research applications. In contrast, full-length fusA protein encompasses the complete amino acid sequence of the native protein, including all structural and functional domains.
The full-length protein is essential for studies requiring all domains and their cooperative interactions, particularly for functional assays of translocation activity. Partial fusA may be preferred in structural studies focused on specific domains, protein-protein interaction analyses of particular regions, or when expression of the complete protein presents technical challenges. When selecting between partial and full-length fusA for research, it's crucial to consider whether the specific research question requires the complete functional protein or if a particular domain is sufficient.
