Recombinant Rhodobacter sphaeroides Ribosome-recycling factor (frr)

What is Rhodobacter sphaeroides Ribosome-recycling factor (frr)?

Rhodobacter sphaeroides Ribosome-recycling factor (frr) is a protein essential for bacterial translation, specifically in the final stage of protein synthesis. It functions to disassemble the post-termination ribosomal complex after translation termination, allowing ribosomes, mRNA, and tRNA to be recycled for subsequent rounds of translation. In Rhodobacter sphaeroides strain ATCC 17029/ATH 2.4.9, the RRF protein consists of 188 amino acids and has been assigned the UniProt accession number A3PJF7 .

What functional domains are present in Rhodobacter sphaeroides RRF?

Based on homology with other bacterial RRFs, the Rhodobacter sphaeroides RRF likely contains:

• Ribosome binding domains that interact with specific regions of the 50S subunit

• Motifs that interact with elongation factor G

• Regions involved in promoting conformational changes in the ribosome

These functional elements work together to ensure efficient recycling of translation components.

How can I assess the purity and activity of recombinant Rhodobacter sphaeroides RRF?

For research applications, recombinant Rhodobacter sphaeroides RRF should be assessed for:

• Purity: SDS-PAGE analysis (>85% purity is typically suitable for most research applications)

• Activity: In vitro ribosome splitting assays measuring the ability of RRF, in conjunction with EF-G and GTP, to dissociate 70S ribosomes into 30S and 50S subunits

• Structural integrity: Circular dichroism spectroscopy to confirm proper folding

What ribosome profiling approaches can be used to study Rhodobacter sphaeroides RRF function?

Ribosome profiling offers powerful insights into RRF function in vivo. Based on studies in E. coli , an effective experimental design would include:

• Establish a conditional RRF depletion system in Rhodobacter sphaeroides

• Harvest cells at various timepoints after initiating depletion

• Prepare ribosome profiling libraries with both standard and high-salt (1M NaCl) lysis buffers

• Deep sequencing of ribosome-protected mRNA fragments

• Bioinformatic analysis to map ribosome positions across the transcriptome

This approach allows for observation of:

• Accumulation of ribosomes at stop codons

• Queuing patterns of elongating ribosomes upstream of stop codons

• Distribution of ribosome density in 3'-UTRs

• Differential effects of high-salt conditions on various ribosomal complexes

How can the interaction between Rhodobacter sphaeroides RRF and other translation factors be investigated?

Several methodological approaches can be employed:

In vitro interaction assays:

• Surface plasmon resonance (SPR) to measure binding kinetics with purified factors

• Pull-down assays using tagged RRF to identify interaction partners

• Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

Structural studies:

• Cryo-electron microscopy of RRF-ribosome complexes

• X-ray crystallography of RRF in complex with binding partners

• Hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

Genetic approaches:

• Site-directed mutagenesis to identify critical residues for interaction

• Suppressor mutation analysis to identify functional relationships

What are the effects of high-salt conditions on ribosomal complexes containing RRF?

High-salt conditions provide valuable insights into the nature of ribosomal complexes. Studies in E. coli have demonstrated that:

• Post-termination complexes (post-TCs) at stop codons are destabilized by high salt (1M NaCl)

• Elongating ribosomes with intact peptidyl-tRNA remain stable in high salt

• Ribosome density in 3'-UTRs is significantly reduced under high-salt conditions

These differential effects can be exploited to distinguish between actively translating ribosomes and post-TCs that have failed to be recycled, providing a valuable tool for studying RRF function.

How does RRF depletion affect translation and cellular physiology?

Based on studies in E. coli, RRF depletion has several significant effects that would likely be mirrored in Rhodobacter sphaeroides :

Interestingly, RRF depletion in E. coli does not significantly alter the ratio of ribosome density on neighboring genes in polycistronic transcripts, suggesting that re-initiation after recycling is not a widespread mechanism .

How can the role of Rhodobacter sphaeroides RRF in cellular stress responses be investigated?

The potential role of RRF in stress adaptation can be investigated through:

Transcriptomic and proteomic approaches:

• Ribosome profiling under various stress conditions with and without RRF depletion

• RNA-seq to profile gene expression changes

• Proteomics to identify proteins affected by RRF depletion during stress

Physiological assays:

• Survival rates under oxidative, temperature, or nutrient stress

• Recovery kinetics after stress exposure

• Polysome profiling to examine translation status during stress

Molecular dynamics:

• Localization studies using fluorescently tagged RRF

• Analysis of potential stress-induced modifications of RRF

Can Rhodobacter sphaeroides extract properties be attributed to RRF activity?

Rhodobacter sphaeroides extracts have been shown to have significant biological activities, including antioxidant effects and promotion of cell growth . While the search results don't directly connect these properties to RRF activity, it raises interesting research questions:

• Does RRF contribute to the beneficial effects of Rhodobacter sphaeroides extracts?

• Could RRF have functions beyond translation that contribute to stress resistance?

• Does the robust translational machinery of Rhodobacter sphaeroides, including RRF, enable the production of bioactive compounds?

Studies have shown that Rhodobacter sphaeroides extracts can inhibit ROS generation and exert antioxidant effects in human cells . Investigating whether RRF plays a role in these effects represents an interesting avenue for future research.

How does Rhodobacter sphaeroides RRF compare to RRF in other bacterial species?

Comparative analysis can reveal evolutionary adaptations in RRF function:

Sequence alignment and structural modeling would reveal conserved residues and potential functional differences between Rhodobacter sphaeroides RRF and RRFs from other bacterial species.

What insights from E. coli RRF studies can be applied to understanding Rhodobacter sphaeroides RRF?

Studies of E. coli RRF using ribosome profiling have revealed several key insights :

• Accumulation of post-TCs at stop codons when RRF is depleted

• Movement of post-TCs into 3'-UTRs over time

• Differential stability of ribosomal complexes in high salt

• Upregulation of rescue factors like ArfA in response to RRF depletion

These findings provide a valuable framework for investigating similar phenomena in Rhodobacter sphaeroides, while also exploring potential differences arising from its distinct physiology and environmental adaptations.

How might RRF function differ in photosynthetic bacteria like Rhodobacter sphaeroides?

Rhodobacter sphaeroides, as a photosynthetic bacterium, faces unique physiological demands that may influence RRF function:

• Adaptation to changing light conditions requiring rapid translational responses

• Coordination with photosynthetic apparatus assembly

• Potential interaction with additional regulatory factors

Research exploring these aspects could reveal novel aspects of RRF function specific to photosynthetic bacteria.

What are the potential applications of understanding Rhodobacter sphaeroides RRF in biotechnology?

Understanding RRF function could contribute to:

• Optimization of protein production in bacterial expression systems

• Development of new antibacterial targets

• Enhanced production of valuable compounds in Rhodobacter sphaeroides

• Improved strain engineering for biotechnological applications

Rhodobacter sphaeroides is known to produce valuable compounds including antioxidants, coenzyme Q10, and carotenoids . Manipulating RRF function could potentially enhance these capabilities.

How can systems biology approaches contribute to understanding the global impact of RRF function?

Integrative approaches combining multiple data types could provide comprehensive insights:

• Multi-omics integration (transcriptomics, proteomics, metabolomics)

• Network analysis of translation-related factors

• Mathematical modeling of ribosome dynamics

• Evolutionary analysis across bacterial species

These approaches would place RRF function in the broader context of cellular physiology and adaptation.