Recombinant Acidovorax ebreus Peptide chain release factor 1 (prfA)

How does the structure of Acidovorax ebreus prfA compare to similar proteins in other bacteria?

Based on comparative genomics, A. ebreus prfA likely shares structural similarity with other bacterial peptide chain release factors. These proteins typically contain domains for stop codon recognition and peptidyl-tRNA hydrolysis. In other bacteria, RF1 and RF2 exhibit significant sequence homology, suggesting similar tertiary structures despite their different stop codon specificities . Detailed structural studies specifically on A. ebreus prfA would require X-ray crystallography or cryo-EM analysis to confirm these structural predictions.

What genomic features characterize the prfA gene in Acidovorax ebreus?

The prfA gene in Acidovorax ebreus is part of its completed genome sequence. While the search results don't provide specific details about the prfA gene locus, the complete genome of A. ebreus strain TPSY has been sequenced, revealing an organism optimized for survival in complex environmental systems . Genomic analysis using platforms like Anvi'o 6.2 would allow identification and characterization of the prfA gene within the context of the entire A. ebreus genome .

What are the optimal expression systems for recombinant Acidovorax ebreus prfA?

For recombinant expression of A. ebreus prfA, E. coli is likely the preferred heterologous expression system, as demonstrated with other A. ebreus proteins. For example, the recombinant UPF0060 membrane protein from A. ebreus was successfully expressed in E. coli with an N-terminal His-tag . When expressing A. ebreus prfA, researchers should consider:

What purification strategies are most effective for recombinant A. ebreus prfA?

Based on protocols for similar bacterial proteins, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing step: Size exclusion chromatography to achieve high purity

For buffer optimization, consider using Tris/PBS-based buffer with 6% trehalose at pH 8.0, similar to conditions used for other A. ebreus proteins . Adding glycerol (5-50% final concentration) helps maintain stability during storage, with 50% being commonly used for long-term storage at -20°C/-80°C .

How can researchers optimize storage conditions for recombinant A. ebreus prfA?

To maintain stability and functionality of purified recombinant A. ebreus prfA:

• Store at -20°C/-80°C upon receipt

• Perform aliquoting for multiple use to avoid repeated freeze-thaw cycles

• Consider adding glycerol to a final concentration of 50%

• For working stocks, store aliquots at 4°C for up to one week

• Avoid repeated freezing and thawing as this can significantly reduce protein activity

What assays can be used to measure A. ebreus prfA activity in vitro?

To assess the functional activity of recombinant A. ebreus prfA, researchers can employ several in vitro assays:

• Peptidyl-tRNA hydrolysis assay: Measure the release of radiolabeled or fluorescently labeled peptides from purified peptidyl-tRNA substrates

• Translation termination efficiency assay: Utilize reconstituted in vitro translation systems with reporter constructs containing specific stop codons

• Ribosome binding assay: Assess direct binding between purified A. ebreus prfA and bacterial ribosomes using techniques such as surface plasmon resonance or filter binding assays

These assays would need to be adapted from protocols established for other bacterial release factors, as specific A. ebreus prfA activity assays are not described in the search results.

How does the codon specificity of A. ebreus prfA compare to other bacterial species?

Based on knowledge of other bacterial release factors, A. ebreus prfA likely recognizes UAG and UAA stop codons, similar to RF1 in E. coli . To experimentally determine codon specificity, researchers could:

• Perform in vitro translation termination assays using mRNAs with different stop codons

• Create genetic complementation studies in RF-deficient bacterial strains

• Conduct mutational analysis of the codon recognition domain

• Use bioinformatic approaches to compare the recognition domains with well-characterized release factors

What is known about the regulation of prfA expression in Acidovorax ebreus?

While specific information about A. ebreus prfA regulation is not provided in the search results, insights can be drawn from studies on peptide chain release factors in other bacteria. In E. coli, RF2 (a related release factor) is regulated by a unique autogenous mechanism involving an in-frame UGA stop codon that requires a +1 frameshift for expression . This results in tight regulation of RF2 levels, which are maintained at low concentrations relative to other translation factors .

Similar regulatory mechanisms might exist for A. ebreus prfA, though experimental verification would be necessary. Techniques to study prfA regulation could include:

• Reporter gene fusions to monitor expression levels

• Quantitative RT-PCR to measure transcript abundance under different conditions

• Proteomic analysis to determine protein levels in various growth phases

How might environmental factors influence A. ebreus prfA function?

Acidovorax ebreus is an anaerobic iron-oxidizing bacterium, optimized for survival in complex environmental systems . Environmental factors that might influence prfA function include:

• Oxygen levels: As an anaerobic organism, A. ebreus likely has adaptations in translation termination efficiency under varying oxygen concentrations

• Iron availability: Given its iron-oxidizing capabilities, iron concentration may affect global gene expression, potentially including prfA

• Nitrogen sources: A. ebreus is nitrate-dependent , suggesting potential regulatory links between nitrogen metabolism and translation efficiency

Researchers could investigate these environmental influences through comparative proteomic and transcriptomic analyses under varying growth conditions.

What structural analysis techniques are most suitable for A. ebreus prfA?

For comprehensive structural characterization of A. ebreus prfA, researchers should consider:

• X-ray crystallography: To obtain high-resolution structures, potentially in complex with ribosomal components

• Cryo-electron microscopy: Particularly valuable for visualizing prfA in the context of the ribosomal termination complex

• Hydrogen/deuterium exchange mass spectrometry: To identify dynamic regions and conformational changes upon substrate binding

• Small-angle X-ray scattering (SAXS): For solution-state structural information

• Nuclear magnetic resonance (NMR): For studying dynamics and smaller domains of the protein

These approaches could reveal insights similar to those found for other bacterial release factors or regulatory proteins like Listeria PrfA, where crystal structure analysis has been crucial for understanding function .

How can researchers investigate interactions between A. ebreus prfA and other translation factors?

To study the protein-protein interaction network of A. ebreus prfA, researchers could employ:

• Pull-down assays: Using tagged recombinant prfA to identify interacting partners

• Yeast two-hybrid or bacterial two-hybrid screens: For systematic identification of protein partners

• Biolayer interferometry or surface plasmon resonance: To measure binding kinetics and affinities

• Crosslinking coupled with mass spectrometry: To capture transient interactions during translation termination

• Fluorescence resonance energy transfer (FRET): For studying interactions in solution or in vivo

These methods could help map the functional interaction network of prfA within the context of the A. ebreus translation machinery.

How can site-directed mutagenesis be applied to study A. ebreus prfA functional domains?

Researchers can use site-directed mutagenesis to systematically analyze structure-function relationships in A. ebreus prfA:

• Target conserved residues identified through sequence alignment with well-characterized release factors

• Focus on domains responsible for:

  • Stop codon recognition

  • Peptidyl-tRNA hydrolysis

  • Ribosome binding

• Create alanine scanning libraries across functional domains

• Design mutations based on predictive structural models

Mutants can be assessed using the functional assays described in section 3.1, allowing correlation of structural features with specific functions.

What insights from other bacterial release factors might apply to A. ebreus prfA research?

Key insights that may be applicable to A. ebreus prfA research include:

• The frameshifting mechanism observed in E. coli RF2 expression occurs at a remarkably high rate of 50%, suggesting sophisticated translational regulation

• Release factors are typically found at low concentrations relative to other translation factors, indicating tight regulation

• The significant sequence homology between RF1 and RF2 in E. coli suggests a common evolutionary origin despite different specificities

• The binding promiscuity observed with regulatory proteins like Listeria PrfA might provide insights into potential moonlighting functions

These concepts could guide experimental design for A. ebreus prfA characterization, especially regarding regulatory mechanisms and evolutionary relationships.

How does A. ebreus prfA fit within the evolutionary context of bacterial release factors?

To explore the evolutionary context of A. ebreus prfA, researchers should consider:

• Conducting phylogenetic analysis across diverse bacterial species

• Comparing sequence conservation patterns in functional domains

• Analyzing selection pressures on different regions of the protein

• Investigating potential horizontal gene transfer events

Tools like Anvi'o 6.2 used for Acidovorax pan-genome analysis could be applied to study prfA evolution across strains . This approach could reveal how prfA contributed to A. ebreus adaptation to its ecological niche as an anaerobic iron-oxidizer.

What can pan-genomic analysis reveal about prfA variation across Acidovorax species?

Pan-genomic analysis, as applied to Acidovorax species in previous studies , could reveal:

• Conservation levels of prfA across different Acidovorax strains

• Presence of paralogous genes or alternative release factors

• Strain-specific variations that might correlate with ecological adaptations

• Co-evolution patterns with interacting translation factors