Recombinant Kineococcus radiotolerans Ribosome-recycling factor (frr)
Product Specs
Target Background
KEGG: kra:Krad_1418
STRING: 266940.Krad_1418
Q&A
Experimental Design for Studying Recombinant Kineococcus radiotolerans Ribosome-recycling Factor (frr)
Q: How should I design an experiment to study the function of recombinant Kineococcus radiotolerans ribosome-recycling factor (frr) in ribosome recycling?
A: To study the function of recombinant Kineococcus radiotolerans ribosome-recycling factor (frr), you can use a combination of biochemical assays and structural studies. First, express and purify the recombinant frr protein using a suitable host like yeast. Then, perform in vitro ribosome recycling assays using purified ribosomes and frr. Analyze the results using techniques such as gel filtration or cryo-electron microscopy to visualize the interaction between frr and ribosomes. Additionally, consider using mutagenesis to study the role of specific residues in frr's function.
Data Analysis and Contradiction Resolution
Q: How do I resolve contradictions in data when comparing the ribosome recycling efficiency of recombinant Kineococcus radiotolerans frr with other bacterial frr proteins?
A: To resolve contradictions, ensure that experimental conditions are consistent across different studies. Check for variations in ribosome preparation, buffer conditions, and temperature. Use statistical analysis to compare results and consider repeating experiments with standardized protocols. Also, examine the structural differences between frr proteins from different species, as these might influence their interaction with ribosomes.
Advanced Research Questions: Structural Insights
Q: What structural insights can be gained from studying the interaction between recombinant Kineococcus radiotolerans frr and ribosomes?
A: Studying the interaction between frr and ribosomes can provide insights into how frr mimics tRNA to facilitate ribosome recycling. Use techniques like cryo-electron microscopy (cryo-EM) or X-ray crystallography to determine the high-resolution structure of the frr-ribosome complex. This can reveal specific contacts between frr and ribosomal elements, such as the A/P site, and how these interactions facilitate ribosome recycling.
Mechanistic Studies: Role of frr in Ribosome Recycling
Q: How does recombinant Kineococcus radiotolerans frr contribute to the mechanistic understanding of ribosome recycling in bacteria?
A: Recombinant Kineococcus radiotolerans frr contributes to understanding ribosome recycling by demonstrating how it interacts with ribosomes to release them from mRNA after translation termination. This process is crucial for maintaining ribosomal availability for new rounds of translation. By studying frr's role, researchers can better understand how bacteria efficiently recycle ribosomes, especially under stress conditions.
Comparative Analysis with Other Bacterial frr Proteins
Q: How does the recombinant Kineococcus radiotolerans frr compare functionally and structurally to frr proteins from other radiation-resistant bacteria like Deinococcus radiodurans?
A: Compare the structural and functional properties of frr from Kineococcus radiotolerans with those from Deinococcus radiodurans by analyzing their sequences, expression levels, and interactions with ribosomes. Use bioinformatics tools to align sequences and predict structural similarities or differences. Functionally, assess how each frr protein affects ribosome recycling efficiency under various conditions, such as oxidative stress.
Methodological Considerations for Protein Expression and Purification
Q: What are the best practices for expressing and purifying recombinant Kineococcus radiotolerans frr protein for structural and functional studies?
A: For optimal expression and purification of recombinant frr, use a suitable expression host like yeast, which can provide high yields of soluble protein. Optimize expression conditions by varying temperature, inducer concentration, and growth time. For purification, use affinity chromatography followed by size exclusion chromatography to achieve high purity (>85%). Store purified protein at -20°C or -80°C with appropriate stabilizers like glycerol to maintain stability.
In Vivo Studies: Translational Coupling and Ribosome Recycling
Q: How can in vivo studies using translational coupling systems help elucidate the role of recombinant Kineococcus radiotolerans frr in ribosome recycling?
A: In vivo studies using translational coupling systems can demonstrate how frr facilitates the release of ribosomes from mRNA after termination, allowing them to re-initiate translation on downstream ORFs. Construct plasmids with specific junction sequences between upstream and downstream ORFs and measure the effect of frr on downstream ORF translation. This approach can provide insights into how frr functions in the context of cellular translation processes.
Synergistic Action with Elongation Factor G (EF-G)
Q: How does recombinant Kineococcus radiotolerans frr synergize with elongation factor G (EF-G) during ribosome recycling?
A: The synergistic action between frr and EF-G involves frr's role in positioning the ribosome for EF-G's action. Frr primes the intersubunit bridge B2a, facilitating EF-G's GTP-dependent activity to split the ribosome into subunits. This coordinated action ensures efficient ribosome recycling and maintains translational capacity. Study this synergy using biochemical assays that measure the efficiency of ribosome splitting in the presence and absence of both factors.
Stress Conditions and Ribosome Recycling
Q: How does recombinant Kineococcus radiotolerans frr contribute to ribosome recycling under stress conditions, such as oxidative stress?
A: Under stress conditions like oxidative stress, frr's role in ribosome recycling becomes critical for maintaining cellular translational capacity. Study how frr's expression and activity are affected by oxidative stress and how it interacts with other stress response mechanisms to ensure ribosome availability. Use techniques like quantitative PCR to measure frr mRNA levels and Western blotting to assess protein stability under stress.
Future Directions in Research
Q: What are potential future research directions for studying recombinant Kineococcus radiotolerans frr in the context of ribosome recycling?
A: Future research should focus on elucidating the structural dynamics of frr-ribosome interactions using advanced techniques like time-resolved cryo-EM. Additionally, explore how frr's function is modulated by post-translational modifications or interactions with other ribosomal factors. Investigate the potential applications of frr in biotechnology, such as enhancing protein production in industrial strains.
Example Data Table: Comparison of frr Proteins from Different Bacteria
| Bacterial Species | frr Sequence Identity | Ribosome Recycling Efficiency | Structural Features |
|---|---|---|---|
| Kineococcus radiotolerans | High similarity to E. coli frr | Efficient recycling under stress | Mimics tRNA structure |
| Deinococcus radiodurans | Moderate similarity to E. coli frr | Enhanced recycling under oxidative stress | Unique interactions with ribosomal elements |
| Escherichia coli | Reference sequence | Baseline recycling efficiency | Well-characterized tRNA-like structure |
Detailed Research Findings: Structural Insights into frr-Ribosome Interaction
The interaction between frr and ribosomes involves frr binding to the A/P site of the ribosome, mimicking the structure of tRNA. This binding facilitates the release of ribosomes from mRNA after translation termination, allowing them to recycle for new rounds of translation. Structural studies using cryo-EM have revealed that frr's orientation on the ribosome is upside-down compared to tRNA, but it still effectively primes the ribosome for the action of elongation factor G (EF-G) during recycling .
