Recombinant Nitrosomonas europaea Peptide chain release factor 3 (prfC), partial

What is peptide chain release factor 3 (prfC) and what is its function in bacteria?

In vitro and in vivo studies demonstrate that RF3 deficiency leads to increased sensitivity to errors in protein synthesis, particularly when combined with drugs or mutations that increase miscoding events . This suggests that RF3 works as a surveillance mechanism during translation to ensure accurate protein production.

What is the genomic context of prfC in Nitrosomonas europaea?

Nitrosomonas europaea contains a single circular chromosome of 2,812,094 bp with approximately 2,460 protein-encoding genes averaging 1,011 bp in length . While the search results don't specifically identify the prfC gene's exact location in N. europaea, the genome is distributed into two unequal replichores with genes evenly distributed around the genome (approximately 47% transcribed from one strand and 53% from the complementary strand) .

Given the essential nature of protein synthesis quality control, the prfC gene would be expected to be among the core genes conserved in this organism, though potentially with unique adaptations related to N. europaea's specialized metabolism as an ammonia oxidizer.

How does prfC function differ in Nitrosomonas europaea compared to other bacteria?

While the search results don't provide direct comparative data specific to N. europaea prfC, research on other bacterial systems indicates that RF3 function can have species-specific characteristics. In general, RF3 works with release factors RF1 and RF2 during translation termination, but its primary role appears to be in quality control .

For N. europaea, an obligate chemolithoautotroph with a specialized metabolism centered around ammonia oxidation, the quality control function of RF3 might be particularly important for maintaining proteome integrity under the stressful conditions of ammonia oxidation, which can generate reactive nitrogen species.

What are the optimal conditions for expressing recombinant N. europaea prfC in heterologous systems?

Based on research with N. europaea and other bacterial expression systems, the following conditions are recommended:

Expression System Parameters:

When working with N. europaea proteins, it's important to consider that this organism grows optimally at 30°C in the dark with gentle agitation (175 rpm) . These conditions can be adapted for heterologous expression systems to maintain protein folding similar to native conditions.

What purification strategies are most effective for isolating recombinant N. europaea prfC?

For purifying recombinant N. europaea prfC, a multi-step chromatography approach is typically most effective:

Recommended Purification Protocol:

• Affinity chromatography (His-tag or other fusion tag) as initial capture step

• Ion exchange chromatography for intermediate purification

• Size exclusion chromatography as polishing step

Buffer composition should be optimized specifically for prfC stability, typically including:

• 50 mM phosphate buffer (pH 7.0-7.5)

• 100-300 mM NaCl

• 5% glycerol as stabilizer

• 1 mM DTT to maintain reduced cysteines

These conditions are consistent with phosphate buffer systems used in N. europaea research (9.2 mM KH₂PO₄ and 10.7 mM K₂HPO₄, pH 7) for enzyme activity assays .

How can researchers verify the functionality of recombinant N. europaea prfC?

To verify functionality of recombinant N. europaea prfC, researchers should consider complementation assays in prfC-deficient systems:

Functional Verification Methods:

• Complementation assay: Transform the recombinant prfC into a Δprfc strain to restore wild-type phenotype. Successful complementation would rescue growth defects observed in prfC-deficient strains under stress conditions, such as exposure to streptomycin or other miscoding agents .

• In vitro translation assay: Use a cell-free translation system to assess the ability of purified recombinant prfC to enhance translation accuracy and reduce premature termination events.

• Streptomycin sensitivity test: Compare growth of wild-type, ΔprfC, and complemented strains in media containing increasing concentrations of streptomycin. The complemented strain should show restoration of wild-type tolerance levels .

How does prfC deletion affect N. europaea's response to environmental stressors?

While specific data for N. europaea is not provided in the search results, research with other bacterial systems shows that prfC deletion significantly impacts stress responses. Studies indicate that RF3 is critical for cells under various stress conditions .

For N. europaea, which encounters environmental stresses including fluctuating ammonia concentrations and exposure to reactive nitrogen species, prfC likely plays a crucial role in maintaining proteome integrity. In particular:

• Nitrosative stress response: As an ammonia oxidizer that produces NO as an intermediate, N. europaea requires mechanisms to handle nitrosative stress. RF3-mediated quality control likely helps maintain protein integrity under these conditions.

• Growth under limiting conditions: prfC deletion strains would likely show greater sensitivity to growth-limiting conditions common in N. europaea's environmental niches.

• Adaptation to pH changes: N. europaea produces nitrite, which can acidify its environment; RF3 may be important for maintaining protein synthesis fidelity under changing pH conditions.

What is the relationship between prfC function and nitrification efficiency in N. europaea?

While direct experimental evidence is not provided in the search results, we can reasonably hypothesize connections based on known functions:

The nitrification process in N. europaea involves ammonia oxidation to hydroxylamine by ammonia monooxygenase (AMO), followed by hydroxylamine oxidation to nitrite . This process:

• Generates reactive nitrogen species

• Requires precise regulation of enzyme expression

• Involves complex electron transport systems

RF3's quality control function during translation is likely critical for maintaining the integrity of these enzyme systems. Defects in protein synthesis fidelity would compromise the efficiency of the nitrification machinery. Particularly, the integrity of the AMO complex and electron transport components would be sensitive to translational errors.

A potential experimental approach to investigate this relationship would be to construct a conditional prfC mutant in N. europaea and measure nitrification rates under various conditions.

How does the presence of recombinant prfC affect the sensitivity of N. europaea to antibiotics that target protein synthesis?

Based on research with other bacteria, prfC deletion makes cells significantly more sensitive to antibiotics that increase miscoding, such as streptomycin . For N. europaea specifically:

• Increased sensitivity: Wild-type N. europaea would be expected to tolerate higher concentrations of streptomycin and other aminoglycosides compared to prfC deletion strains.

• Complementation effect: Introduction of recombinant prfC should restore antibiotic tolerance to levels similar to wild-type.

The experimental data on E. coli shows that a ΔprfC strain completely failed to grow at antibiotic concentrations tolerated by the wild-type strain, and this phenotype was rescued by introducing plasmid-borne prfC . Similar effects would be expected in N. europaea.

Furthermore, in error-prone (ram) strains, the deletion of prfC was particularly detrimental to growth, suggesting an additive effect when translation accuracy is already compromised .

What structural features of N. europaea prfC are critical for its function in quality control during translation?

While the search results don't provide specific structural information for N. europaea prfC, based on research with RF3 from other bacteria, several key structural features are likely critical:

• G-domain: The GTPase domain is essential for RF3 function, as it undergoes conformational changes upon GTP binding and hydrolysis.

• Domain interactions: The interaction between RF3 and the ribosome involves multiple domains that recognize specific ribosomal features and other translation factors.

• Species-specific adaptations: N. europaea as an ammonia oxidizer may have specific structural adaptations in prfC to function optimally under its unique metabolic conditions.

A comprehensive structural analysis of recombinant N. europaea prfC using X-ray crystallography or cryo-EM would be valuable to identify specific features that may differ from well-characterized RF3 proteins in other bacteria.

How does prfC interact with the nitrosative stress response system in N. europaea?

N. europaea produces NO during ammonia oxidation and possesses specific systems for dealing with nitrosative stress, including a functional nitric oxide reductase encoded by the norCBQD gene cluster . The potential interaction between prfC and this system is multifaceted:

• Quality control for NO-handling proteins: RF3 likely ensures proper synthesis of proteins involved in NO detoxification, including the nor gene products.

• Response to protein damage: Nitrosative stress can damage proteins, potentially increasing the burden on quality control systems like RF3.

• Coordinated regulation: The expression of prfC may be coordinated with stress response systems to maintain cellular function under challenging conditions.

Experimental approaches to investigate this interaction could include analyzing changes in nor gene expression and function in prfC mutant strains, particularly under conditions of elevated NO production.

What is the role of prfC in premature termination events during protein synthesis in N. europaea?

Research indicates that RF3 affects premature termination events in protein synthesis. Specifically:

• Reduced premature termination in prfC deletion strains: Studies show that premature termination is significantly reduced in ΔprfC strains, suggesting that RF3 normally promotes termination at certain error sites .

• Quality control mechanism: This likely represents a quality control mechanism where RF3 helps terminate translation when errors are detected, preventing the production of potentially harmful partial proteins.

• Balance between quality and quantity: This function creates a balance between protein synthesis accuracy (quality) and efficiency (quantity).

In N. europaea, this quality control function would be particularly important for maintaining the integrity of key metabolic enzymes involved in ammonia oxidation and energy generation.

What are the current methodological challenges in studying recombinant N. europaea prfC?

Several methodological challenges exist in studying recombinant N. europaea prfC:

• Expression system selection: N. europaea has specialized metabolic adaptations that may affect protein folding and function when expressed in standard laboratory hosts like E. coli.

• Functional assays: Developing specific assays to measure RF3 activity in the context of N. europaea's unique metabolism requires specialized approaches.

• Genetic manipulation: While genetic systems exist for N. europaea, they may be less efficient than those for model organisms, making targeted genetic studies more challenging.

• Protein-protein interaction studies: Identifying N. europaea-specific interaction partners for RF3 requires specialized proteomic approaches given the unique metabolic context.

How can transcriptomic and proteomic approaches enhance our understanding of prfC function in N. europaea?

Multi-omics approaches offer powerful insights into prfC function:

Transcriptomic Applications:

• RNA-seq analysis comparing wild-type and prfC mutant strains can reveal gene expression changes resulting from altered translation quality control.

• Ribosome profiling can identify specific mRNAs most affected by prfC deletion, revealing targets particularly dependent on RF3-mediated quality control.

Proteomic Applications:

• Quantitative proteomics comparing wild-type and prfC mutant strains can identify proteins whose levels are most affected by RF3 deficiency.

• Analysis of protein modifications in prfC mutants could reveal increased error incorporation or premature termination events.

These approaches would be particularly valuable since RF3 deletion has been shown to significantly alter the transcriptome and proteome in other bacterial systems .

What are promising future research directions for understanding the intersection of prfC function and N. europaea's specialized metabolism?

Future research should focus on several promising directions:

• Metabolic integration: Investigating how RF3-mediated quality control is integrated with N. europaea's specialized ammonia oxidation metabolism, particularly under varying ammonia concentrations.

• Environmental adaptation: Exploring how prfC function contributes to N. europaea's adaptation to changing environmental conditions in natural and engineered systems.

• Synthetic biology applications: Developing engineered N. europaea strains with modified prfC to enhance nitrification efficiency or stress tolerance for environmental applications.

• Comparative studies: Comparing prfC function across different ammonia-oxidizing bacteria and archaea to identify conserved and divergent features related to their ecological niches.

• Structure-function relationships: Determining the three-dimensional structure of N. europaea RF3 to identify unique features that may reflect adaptation to its specialized metabolism.