Recombinant Nitrosomonas europaea Translation initiation factor IF-2 (infB), partial

What is the function of Translation initiation factor IF-2 in Nitrosomonas europaea?

Translation initiation factor IF-2 in N. europaea, like in other bacteria, catalyzes the binding of initiator tRNA to the initiating 30S ribosomal subunit and subsequently promotes the formation of the 70S ribosome . This protein is absolutely conserved in all bacteria, highlighting its essential role in translation. IF-2 contains a GTPase domain, and ribosome-stimulated GTP hydrolysis is required for rapid dissociation of the factor from the ribosome following initiation complex formation . Additionally, research has revealed that bacterial IF-2 plays an unexpected role in ribosome assembly and maturation, particularly at low temperatures .

Why is recombinant expression of N. europaea IF-2 challenging?

N. europaea is a slow-growing obligate chemolithoautotroph with cell division taking several days due to the large amounts of ammonia required for energy . This slow growth makes direct purification of native proteins difficult. Additionally, N. europaea has distinct codon usage patterns that may not be optimal for expression in common laboratory hosts like E. coli. When expressing N. europaea proteins recombinantly, researchers typically optimize gene sequences for expression in E. coli, as was done in studies of other N. europaea proteins . The large size of IF-2 (containing multiple domains including G1, G2, G3, C1, and C2 domains) also presents challenges for proper folding and solubility when expressed recombinantly .

What experimental methods are effective for purifying recombinant N. europaea IF-2?

Based on protocols used for other bacterial IF-2 proteins and recombinant N. europaea proteins, the following methods are recommended:

• Affinity chromatography: Expression with a His-tag or GST-tag followed by affinity purification. This approach was used successfully for IF-2 variants in research .

• Optimized expression conditions: Cold-induction (16-18°C) often improves the solubility of large proteins like IF-2.

• Buffer optimization: Including GTP or GDP (1-5 mM) in purification buffers can stabilize the protein's structure.

• Sequential chromatography: Follow affinity purification with ion exchange and size exclusion chromatography for higher purity.

This multi-step purification strategy has proven effective for obtaining functionally active recombinant translation factors for biochemical studies .

How can the functionality of purified recombinant N. europaea IF-2 be verified?

Several assays can verify the functionality of purified recombinant IF-2:

• GTPase activity assay: Measure the GTP hydrolysis rate using colorimetric or fluorescent assays. Functional IF-2 should show ribosome-stimulated GTPase activity .

• 30S binding assay: Assess binding to 30S ribosomal subunits using filter binding or fluorescence anisotropy methods.

• Initiator tRNA binding assay: Evaluate the binding of fMet-tRNA using electrophoretic mobility shift assays.

• 70S formation assay: Monitor the ability to promote 50S and 30S subunit joining using light scattering or sucrose gradient ultracentrifugation .

• Thermal shift assay: Determine if the protein binds nucleotides (GTP/GDP) by measuring changes in thermal stability upon nucleotide binding .

How does the cold stress response affect IF-2 expression and function in N. europaea?

Cold stress in bacteria, including N. europaea, results in increased synthesis of translation initiation factors IF-1, IF-2, and IF-3, with the IFs/ribosome stoichiometric ratio increasing approximately 3-fold during the first hours of cold adaptation . Research has revealed that the extra copies of IF-2 made after cold stress are associated with immature ribosomal subunits together with at least nine other proteins involved in assembly and/or maturation of ribosomal subunits .

Experimental evidence demonstrates that IF-2 possesses GTPase-associated chaperone activity that can promote protein refolding . Cold-sensitive IF-2 mutations cause the accumulation of immature ribosomal particles, indicating that IF-2 is a GTPase protein that participates in ribosome assembly/maturation, especially at low temperatures .

For N. europaea, which inhabits various environments including soil and wastewater treatment systems where temperature fluctuations occur, this cold-responsive function of IF-2 likely plays a significant role in adaptation to environmental stresses. Researchers investigating N. europaea IF-2 should consider experimental designs that account for temperature effects on its function.

What techniques can be used to investigate the structural dynamics of N. europaea IF-2?

Based on successful approaches with bacterial IF-2 proteins, the following techniques are recommended:

• NMR spectroscopy: This technique has been effectively used to study conformational changes in IF-2 domains upon nucleotide binding. NMR studies of Bacillus stearothermophilus IF-2-G2 revealed large structural rearrangements in this subdomain upon GDP binding .

• X-ray crystallography: Can provide high-resolution structures of individual domains or full-length protein.

• Cryo-electron microscopy: Useful for visualizing IF-2 in complex with ribosomes or tRNA.

• FRET (Förster Resonance Energy Transfer): Can monitor real-time conformational changes during initiation complex formation.

• Hydrogen-deuterium exchange mass spectrometry: Useful for analyzing protein dynamics and conformational changes.

These methods can reveal important structural details about N. europaea IF-2, such as how nucleotide binding affects its conformation and how it interacts with the ribosome and initiator tRNA. Research has shown that in bacteria, GDP-induced rearrangements in the G2 domain are not forwarded toward the fMet-tRNA binding C2 subdomain, suggesting independent mobility between domains .

How might the unique metabolism of N. europaea influence studies of its IF-2 protein?

N. europaea has a unique metabolic profile as an obligate chemolithoautotroph that derives all its energy from the oxidation of ammonia to nitrite . This metabolism presents several considerations for studying its IF-2:

• Growth conditions: When studying IF-2 expression or function in native N. europaea, specialized growth media with ammonia as an energy source and carbon dioxide as a carbon source are required . The slow growth rate (division time of several days) must be factored into experimental timelines .

• Stress responses: N. europaea has developed adaptive strategies to cope with periods of ammonia starvation . During recovery from starvation, specific patterns of gene expression are observed, potentially including translation factors.

• Adaptation mechanisms: The bacterium shows adaption and recovery capacities in response to chronic stressors , which might involve translational regulation through IF-2.

• Cold adaptation relevance: As an environmental bacterium found in soil and wastewater, N. europaea experiences temperature fluctuations that may influence IF-2 function, particularly given IF-2's role in cold adaptation .

When studying recombinant N. europaea IF-2, researchers should consider how these metabolic factors might influence protein function in the native organism.

How can site-directed mutagenesis be used to investigate functional domains of N. europaea IF-2?

Site-directed mutagenesis is a powerful approach for investigating the structure-function relationships of IF-2. Based on research with bacterial IF-2, the following strategy is recommended:

• Target selection: Key residues for mutagenesis include:

  • GTP-binding pocket residues (e.g., equivalent to E571K mutation that inactivates GTPase activity)

  • fMet-tRNA binding residues in the C2 domain

  • Residues at domain interfaces that may affect interdomain communication

• Expression system: Use a complementation system with temperature-sensitive E. coli IF-2 mutants to test functionality of N. europaea IF-2 variants .

• Activity assays: Compare the following activities between wild-type and mutant proteins:

  • GTPase activity (basal and ribosome-stimulated)

  • Binding to initiator tRNA

  • 30S and 50S ribosomal subunit interactions

  • Ribosome assembly/maturation functions

• Structural analysis: Combine with the structural methods described in question 2.2 to understand how mutations affect protein conformation.

Previous research has shown that a single amino acid substitution (E571K) in bacterial IF-2 completely inactivates the GTPase activity , demonstrating the power of targeted mutations for understanding function.

What considerations are important when designing expression systems for recombinant N. europaea IF-2?

When designing expression systems for recombinant N. europaea IF-2, several factors should be considered:

• Codon optimization: N. europaea has different codon usage patterns compared to E. coli. Gene sequence optimization for E. coli expression is recommended, as has been done for other N. europaea proteins .

• Expression vectors: Consider different fusion tags and their effects:

  Tag	Advantages	Disadvantages
  His-tag	Small size, minimal impact on structure	May affect metal-binding properties
  GST-tag	Enhanced solubility	Large size may affect function
  MBP-tag	Greatly enhances solubility	Large size requires removal for some applications

• Expression conditions: The following optimization strategies are recommended:

  • Lower temperature (16-18°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Rich media supplemented with glucose or glycerol

  • Co-expression with chaperones (GroEL/ES)

• Domain-based approach: Consider expressing individual domains (G1, G2, G3, C1, C2) separately, as this approach has been successful in studies of bacterial IF-2 .

• Construct design: Include different versions based on naturally occurring isoforms. Bacterial IF-2 can have multiple isoforms due to alternative translation start sites, with some bacteria having a tandem organization of the IF2N domain .

How can recombinant N. europaea IF-2 be used to study ribosome assembly?

To investigate the role of N. europaea IF-2 in ribosome assembly, the following experimental approaches are recommended:

• In vitro ribosome assembly assays:

  • Incubate purified recombinant IF-2 with ribosomal subunits and monitor assembly using sucrose gradient ultracentrifugation

  • Compare assembly efficiency at different temperatures (37°C vs. low temperature)

  • Assess the impact of GTP, GDP, or non-hydrolyzable GTP analogs

• Complementation studies:

  • Express N. europaea IF-2 in E. coli strains with cold-sensitive IF-2 mutations

  • Measure growth rates and ribosome profiles at low temperatures

• Co-immunoprecipitation experiments:

  • Use tagged recombinant IF-2 to identify interacting partners during ribosome assembly

  • Compare interaction profiles at different temperatures

• Ribosome maturation analysis:

  • Monitor the ability of IF-2 to facilitate conversion of immature ribosomal particles to mature subunits

  • Analyze ribosomal RNA processing in the presence/absence of functional IF-2

Research has shown that bacterial IF-2 is associated with immature ribosomal subunits together with at least nine other proteins involved in assembly/maturation of ribosomal subunits, particularly after cold stress .

What proteomic approaches can identify IF-2 interaction partners in N. europaea?

Several proteomic approaches can identify interaction partners of N. europaea IF-2:

• Affinity purification coupled to mass spectrometry (AP-MS):

  • Express tagged recombinant IF-2 in N. europaea or E. coli

  • Purify complexes using affinity chromatography

  • Identify co-purifying proteins by mass spectrometry

• Crosslinking mass spectrometry (XL-MS):

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest complexes and identify crosslinked peptides by MS/MS

  • Map interaction interfaces between IF-2 and partners

• Proximity labeling:

  • Create fusion proteins with proximity-dependent biotin ligase (BioID or TurboID)

  • Express in cells and allow biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

• Hydrogen-deuterium exchange:

  • Compare hydrogen-deuterium exchange profiles of IF-2 alone vs. in complexes

  • Identify protected regions that indicate binding interfaces

Previous research has identified interactions between IF-2 and other proteins involved in ribosome assembly after cold stress , and similar approaches can be applied to N. europaea IF-2.

How does the role of IF-2 in N. europaea compare to its role in other bacteria?

While IF-2's core function in translation initiation is conserved across bacteria, there may be species-specific adaptations, particularly for extremophiles like N. europaea:

• Comparative functional analysis:

  • Express recombinant IF-2 from N. europaea and other bacteria (E. coli, B. subtilis)

  • Compare GTPase activity, fMet-tRNA binding, and ribosome assembly functions

  • Analyze temperature-dependent activities

• Domain swapping experiments:

  • Create chimeric proteins with domains from N. europaea IF-2 and other bacterial IF-2 proteins

  • Test functionality of chimeras in various assays

  • Identify species-specific functional adaptations

• Evolutionary analysis:

  • Compare sequence conservation patterns across bacterial IF-2 proteins

  • Identify positively selected residues that might confer specialized functions

While all bacteria contain only one copy of the IF-2 gene , the protein may have different isoforms due to alternative translation start sites, and the presence of these isoforms varies across species. In E. coli and B. subtilis, both longer and shorter isoforms contain the major functional domains and are biochemically active, but an optimal ratio is required for maximal growth rate .

N. europaea's unique metabolism and environmental adaptations may have influenced the evolution of its IF-2, potentially optimizing it for function in ammonia-oxidizing conditions or during environmental stress responses.

What RNA-sequencing approaches can help study translational regulation by IF-2 in N. europaea?

Several RNA-sequencing approaches can provide insights into translational regulation by IF-2:

• Ribosome profiling (Ribo-seq):

  • Generate genome-wide snapshots of ribosome positions on mRNAs

  • Compare wild-type N. europaea with strains expressing mutant IF-2

  • Identify changes in translation initiation efficiency across the transcriptome

• Translational efficiency analysis:

  • Combine RNA-seq (total mRNA) with Ribo-seq data

  • Calculate translational efficiency for each transcript

  • Identify mRNAs most affected by alterations in IF-2 function

• Start site mapping:

  • Use methods like QTI-seq (quantitative translation initiation sequencing)

  • Map translation initiation sites genome-wide

  • Determine how IF-2 mutations affect start site selection

• Stress response profiling:

  • Compare translational profiles under normal conditions vs. stress (cold, ammonia limitation)

  • Identify stress-responsive mRNAs whose translation depends on IF-2

The role of IF-2 in environmental adaptation is particularly relevant for N. europaea, which must respond to fluctuations in ammonia availability and temperature. During recovery from ammonia starvation, N. europaea shows differential regulation of gene expression , and IF-2 may play a role in translating stress-responsive mRNAs.

What are common issues when working with recombinant N. europaea IF-2 and how can they be addressed?

Based on experience with bacterial translation factors and recombinant proteins from N. europaea, these common issues and solutions are noted:

• Poor expression/solubility:

  • Optimize codon usage for E. coli expression

  • Lower induction temperature (16-18°C)

  • Use solubility-enhancing fusion tags (MBP, SUMO)

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Express individual domains separately

• Loss of GTPase activity:

  • Include GTP or GDP in purification buffers

  • Check for metal ion contamination (especially Zn2+) that can inhibit GTPase activity

  • Verify protein folding by circular dichroism spectroscopy

  • Test the impact of E571K-equivalent mutations known to affect GTPase activity

• Degradation during purification:

  • Include protease inhibitors in all buffers

  • Minimize purification time and keep samples cold

  • Add nucleotides (GTP/GDP) to stabilize the protein

• Poor binding to ribosomes/tRNA:

  • Verify ribosome quality (contamination with RNases can degrade rRNA)

  • Ensure correct folding of the C2 domain responsible for fMet-tRNA binding

  • Optimize buffer conditions (salt, pH) for binding assays

How can structural analysis guide the design of functional studies for N. europaea IF-2?

Structural analysis can provide valuable insights for functional studies of N. europaea IF-2:

• Domain organization analysis:

  • Use protein prediction tools to identify domain boundaries

  • Design constructs for expressing individual domains

  • Create domain deletion variants to test function

• Nucleotide-binding pocket analysis:

  • Identify key residues in the GTP-binding pocket

  • Create mutations that alter GTP binding or hydrolysis

  • Test the impact on ribosome binding and translation initiation

• Interface mapping:

  • Identify interdomain interfaces that might regulate conformational changes

  • Target these interfaces with mutations to test domain communication

  • NMR studies of bacterial IF-2 have shown that the G2-G3-C1 regions undergo reorganization upon GDP binding

• tRNA-binding region analysis:

  • Identify residues in the C2 domain likely involved in fMet-tRNA binding

  • Create mutations to test binding specificity

  • Compare with known structures of bacterial IF-2 C2 domains

Research has shown significant differences between bacterial IF-2 and its archaeal/eukaryotic homologs. While bacterial IF-2's C2 domain is crucial for initiator-tRNA binding, in archaea this function is performed by a different factor . This highlights the importance of studying bacterial-specific features of IF-2.

What factors should be considered when establishing in vitro translation systems with N. europaea components?

When establishing in vitro translation systems incorporating N. europaea components such as recombinant IF-2, consider:

• Buffer composition optimization:

  • Test different pH values (typically 7.0-8.0)

  • Optimize Mg2+ concentration (5-20 mM)

  • Determine optimal K+ or NH4+ concentrations (50-200 mM)

  • Include spermidine (0.5-2 mM) to stabilize RNA structures

• Source of other components:

  • Decide whether to use N. europaea ribosomes or heterologous ribosomes

  • Consider homologous vs. heterologous tRNAs and aminoacyl-tRNA synthetases

  • Test compatibility of N. europaea IF-2 with ribosomes from other species

• Temperature considerations:

  • Determine optimal temperature (N. europaea prefers 20-30°C)

  • Test activity at multiple temperatures to assess cold adaptation functions

• Energy regeneration system:

  • Include ATP/GTP regeneration systems (creatine phosphate/kinase or phosphoenolpyruvate/kinase)

  • Optimize nucleotide concentrations (typically 1-2 mM GTP, 2-5 mM ATP)

• mRNA considerations:

  • Design mRNAs with appropriate Shine-Dalgarno sequences

  • Consider potential translational pausing due to Shine-Dalgarno-like sequences

The unique metabolism of N. europaea may have selected for specific adaptations in its translation machinery, making these optimization steps particularly important when working with its components.

How might studying N. europaea IF-2 contribute to understanding extremophile adaptation mechanisms?

N. europaea thrives in environments with high ammonia concentrations and can adapt to various stresses, making its IF-2 an interesting target for studying extremophile adaptations:

• Comparative genomics approaches:

  • Compare IF-2 sequences across ammonia-oxidizing bacteria and related nitrifiers

  • Identify conserved substitutions that might represent adaptations to their lifestyle

  • Correlate sequence variations with environmental preferences

• Functional characterization under stress conditions:

  • Test recombinant N. europaea IF-2 activity under various conditions:

    • pH stress (N. europaea tolerates pH 6.0-9.0)

    • Temperature variations (optimal 20-30°C)

    • High ammonia concentrations

    • Oxygen limitation (N. europaea can perform nitrifier denitrification under oxygen-limited conditions)

• Cold adaptation studies:

  • Compare the cold response of N. europaea IF-2 with that of mesophilic bacteria

  • Investigate whether N. europaea IF-2 has enhanced chaperone activity at low temperatures

  • Test complementation of cold-sensitive E. coli IF-2 mutants with N. europaea IF-2

N. europaea has adapted to a variety of environmental stresses, and understanding how its translation machinery, particularly IF-2, contributes to these adaptations could provide insights into extremophile biology and potential biotechnological applications.

What potential biotechnology applications might emerge from research on N. europaea IF-2?

Research on N. europaea IF-2 could lead to several biotechnology applications:

• Protein expression optimization:

  • Develop improved expression systems for difficult-to-express proteins

  • Utilize IF-2's chaperone activity to enhance proper folding of recombinant proteins

  • Engineer IF-2 variants with enhanced cold-adaptation properties for low-temperature protein production

• Bioremediation enhancement:

  • Improve the efficiency of N. europaea in wastewater treatment and bioremediation applications

  • Engineer strains with optimized translation machinery for better performance in environmental applications

  • N. europaea is already known for its ability to degrade various pollutants including halogenated organic compounds

• Antimicrobial development:

  • Target bacterial-specific features of IF-2 for antimicrobial development

  • Bacterial IF-2 has structural and functional differences from its eukaryotic homologs , making it a potential antibiotic target

• Biosensors:

  • Develop biosensors using N. europaea translation machinery components

  • Create systems responsive to ammonia or pollutants by linking detection to protein synthesis

N. europaea's unique metabolism and environmental adaptability, combined with IF-2's multiple roles in translation initiation and ribosome assembly, provide numerous opportunities for biotechnological innovation.