Recombinant Escherichia coli Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 (infB) and what is its role in bacterial translation?

Translation Initiation Factor 2 (IF2) is an essential protein in bacterial cells that plays a crucial role in the translation initiation process. Its primary function is to deliver the initiator formylmethionyl-transfer RNA (fMet-tRNAfMet) to the ribosome pre-initiation complex . This delivery is a critical step that allows the ribosome to correctly position at the start codon of mRNA. IF2 subsequently hydrolyzes its bound GTP, which enables the release of fMet-tRNAfMet into the P-site of the ribosome, allowing the translation machinery to transition from initiation to elongation phase . Without functional IF2, bacterial cells cannot efficiently initiate protein synthesis, making it an essential factor for survival.

What are the key structural domains of E. coli IF2?

E. coli IF2 consists of several important structural domains:

• N-terminal domain: Contains a large intrinsically disordered region (IDR) that varies across bacterial species

• Central domain: Contains the GTP-binding domain responsible for GTPase activity

• C-terminal domain: Contains regions for tRNA and fMet binding

The full-length E. coli IF2 (α-isoform) contains approximately 320 amino acids of intrinsically disordered regions, accounting for about 36% of the total protein . Interestingly, E. coli cells produce three protein isoforms of IF2 with distinct regions of the IDR truncated, though all isoforms contain the functional GTP, tRNA, and fMet binding domains required for translation initiation activity .

AlphaFold structural predictions have been used to annotate the domain structure of IF2, providing insights into the relationship between structure and function that were previously difficult to obtain due to the partially disordered nature of the protein .

How can recombinant E. coli IF2 be efficiently expressed and purified?

An optimized methodology for the high-yield expression and purification of E. coli IF2 involves:

• Cloning the INFB gene into a heat-inducible runaway plasmid (such as pCP40)

• Transforming the construct into an appropriate E. coli strain (e.g., C600 [pCI857])

• Placing INFB expression under the control of a strong promoter (like lambda PL)

• Using a heat-induction protocol: typically 42°C induction followed by expression at 37°C for 2 hours

This system can produce approximately 30 times more IF2 than wild-type cells . Purification can be efficiently achieved through a series of ion exchange liquid chromatography steps:

• Q-Sepharose HP column

• MonoQ column

• MonoS column

This FPLC-based purification protocol yields approximately 5 mg of pure, active IF2 from 10 g of overproducing cells in about 8 hours . The purity and activity of the protein should be verified through SDS-PAGE and functional assays such as mant-GTP binding to confirm proper folding .

What experimental approaches can confirm proper folding of recombinant IF2?

To ensure that recombinant IF2 is properly folded and functionally active, researchers should employ multiple complementary approaches:

• Nucleotide binding assays: Testing the protein's ability to bind fluorescent GTP analogs such as mant-GTP can verify the integrity of the G-domain . All properly folded IF2 variants, including those with truncated N-terminal regions, should retain this binding capacity.

• GTPase activity measurements: Functional IF2 should demonstrate GTP hydrolysis in the presence of ribosomes.

• Circular dichroism spectroscopy: This technique can provide information about the secondary structure content of the protein.

• Thermal shift assays: These can assess the thermal stability of the protein and indirectly indicate proper folding.

• fMet-tRNA binding assays: The ability to bind initiator tRNA is a critical functional property that indicates proper folding of the C-terminal domain.

Each of these methods provides different insights into the structural and functional integrity of the recombinant protein, and a combination of approaches is recommended for comprehensive validation.

What is the role of IF2's N-terminal IDR in bacterial cold adaptation?

The N-terminal intrinsically disordered region (IDR) of IF2 plays a crucial role in bacterial adaptation to cold temperatures. Research has revealed that:

• Deletion of the N-terminal IDR results in a cold-sensitive phenotype, with E. coli cells unable to grow at 15°C despite showing normal growth at 37°C .

• The IDR drives phase separation with RNA, forming biomolecular condensates that become more pronounced at cold temperatures .

• These IF2 condensates appear to protect E. coli cells during adaptation to cold conditions, suggesting a protective mechanism for survival during temperature stress .

• Experimental evidence from immunofluorescence studies shows that IF2 is diffuse throughout the cytoplasm at 37°C but forms distinct puncta after cold shock at 4°C for 1 hour .

• The condensation is reversible – when cells are allowed to recover at 37°C for 1 hour after cold shock, the IF2 foci disperse, returning to a diffuse distribution .

This temperature-dependent phase separation behavior appears to be a conserved property across bacterial species and represents a previously unrecognized mechanism for bacterial adaptation to environmental stress.

How does IF2 phase separation contribute to cellular function during cold stress?

IF2 phase separation during cold stress appears to be functionally significant for bacterial survival through several mechanisms:

• Translation regulation: Cold-shock in E. coli leads to a strong reduction in translation . IF2 condensation may be involved in this translation shutdown during adaptation to cold, potentially by sequestering translation factors and/or specific mRNAs.

• Protective condensate formation: IF2 condensates share similarities with cold-induced eukaryotic stress granules , suggesting they may play an analogous protective role across domains of life.

• Selective protein synthesis: The condensates might help prioritize the translation of cold-shock proteins while temporarily halting the synthesis of other proteins.

• RNA protection: By binding RNA molecules within phase-separated droplets, IF2 may protect certain transcripts from degradation during stress conditions.

The data suggests that IF2 condensation is not merely a consequence of cold stress but actively contributes to cellular adaptation. This is supported by the observation that bacteria without the N-terminal IDR of IF2 grow poorly at cold temperatures .

Table 1: Comparison of IF2 behavior under different temperature conditions

In vitro methods:

• Turbidity assays: Measuring solution turbidity at 350 nm can quantify phase separation of purified IF2 with RNA .

• Fluorescence microscopy: Using fluorescently labeled IF2 and RNA to directly visualize droplet formation.

• Salt and RNase sensitivity tests: Treating IF2-RNA droplets with high salt (500 mM KCl) or RNase A can confirm the electrostatic nature of interactions and RNA dependency .

• Temperature-controlled experiments: Comparing droplet formation at different temperatures (e.g., 4°C vs. 37°C) to assess cold-enhancement of phase separation .

In vivo methods:

• Immunofluorescence: Using strains with tagged IF2 (such as infB-SPA) and antibodies to visualize IF2 localization before and after cold shock .

• Fluorescent protein fusions: Creating GFP-tagged IF2 to monitor localization in living cells.

• Cold shock and recovery experiments: Subjecting cells to cold shock (e.g., 4°C for 1 hour) followed by recovery at normal temperature (37°C for 1 hour) to demonstrate the reversibility of condensate formation .

• Colocalization studies: Using fluorescent markers for other cellular components to identify what molecules co-segregate with IF2 condensates.

When designing these experiments, it's crucial to verify that tagging or modifications don't interfere with IF2 function. Growth rate comparison between wild-type and tagged strains should be performed to ensure the tag doesn't impact IF2 function .

How do different nucleotide binding states affect IF2 structure and phase separation properties?

The nucleotide binding state of IF2 significantly influences its structure and phase separation behavior:

• Conformational changes: Studies with Thermus thermophilus IF2 have shown that the protein adopts a compact state when bound to GDP, and an extended conformation when no nucleotide is bound .

• Effect on phase separation: Experimental data indicates that in the presence of GTP, E. coli IF2 shows a slight reduction in condensation compared to the nucleotide-free state .

• Condensate morphology: Both GDP and GTP appear to affect the physical properties of IF2 condensates, causing them to have irregular shapes and clump together, suggesting potential maturation into aggregate-like structures .

• Functional implications: The observation that nucleotide-free IF2 forms the most regular, spherical droplets may indicate a relationship between IF2's functional cycle and its phase separation properties.

These findings suggest that GTP binding and hydrolysis may serve as regulatory mechanisms for IF2 phase separation in vivo, potentially linking translation activity to condensate formation. For experimental studies focused on droplet formation, performing experiments in the absence of nucleotides is recommended to maintain spherical condensate morphology .

How does IF2 structure and function vary across bacteria adapted to different temperature niches?

IF2 proteins from bacteria adapted to different temperature niches show significant variations that reflect their environmental adaptations:

• IDR length variations: Analysis of IF2 sequences across bacteria reveals that thermophiles (heat-loving bacteria) have significantly reduced IDR lengths compared to mesophiles and psychrophiles (cold-loving bacteria) . This suggests that the IDR may be disadvantageous at high temperatures but beneficial in cold environments.

• Phase separation tendency: Predictive algorithms like deePhase and FuzDrop indicate varying propensities for phase separation across species, with psychrophiles generally showing higher phase separation scores .

• Sequence conservation: Despite low sequence identity in the IDR regions (e.g., only 40.8% amino acid identity between E. coli and C. crescentus IF2), the phase separation capability appears to be functionally conserved .

• Experimental validation: Both E. coli (mesophile) and C. crescentus IF2 proteins have been demonstrated to phase separate with RNA, with the process enhanced by cold temperature . This suggests conservation of this property despite sequence divergence.

These variations likely represent evolutionary adaptations to different thermal environments, with the IDR potentially serving as a key adaptation mechanism for bacteria living in variable or cold temperatures.

What experimental strategies can identify the RNA composition of cold-induced IF2 condensates?

Understanding the RNA components of IF2 condensates is crucial for elucidating their functional role. Several experimental approaches can be employed:

• RNA immunoprecipitation followed by sequencing (RIP-seq):

  • Use tagged IF2 (e.g., IF2-SPA) to pull down associated RNAs after cold shock

  • Sequence the bound RNA population to identify enriched transcripts

  • Compare with control samples (normal temperature, IF2ΔNTD variant)

• Proximity labeling coupled with RNA identification:

  • Fuse IF2 to a proximity labeling enzyme (e.g., APEX2)

  • Activate labeling during cold shock to tag nearby RNAs

  • Purify and identify labeled RNAs by sequencing

• FISH combined with IF2 immunofluorescence:

  • Perform fluorescence in situ hybridization for candidate RNAs

  • Co-stain for IF2 using immunofluorescence

  • Assess colocalization in cold-shocked cells

• In vitro reconstitution with defined RNAs:

  • Test phase separation of purified IF2 with different RNA species

  • Compare total RNA, rRNA, tRNA, and mRNAs for their ability to promote condensation

  • Assess RNA sequence or structural preferences

• Comparative analysis between species:

  • Identify common RNA features that drive phase separation with IF2 across bacterial species

  • Correlate with physiological responses to cold stress

These approaches would help determine whether IF2 condensates contain specific RNAs related to cold adaptation or whether they sequester translation machinery more broadly during stress.

How might IF2 condensates be targeted for antimicrobial development?

The essential role of IF2 in bacterial translation and its specific condensation behavior during cold stress presents unique opportunities for antimicrobial development:

• Targeting the N-terminal IDR: The lamotrigine example demonstrates that small molecules binding to IF2's N-terminal IDR can selectively inhibit bacterial growth in cold conditions . This suggests that compounds disrupting phase separation could be effective antimicrobials with novel mechanisms of action.

• Temperature-specific antibiotics: Compounds that interfere with IF2 condensation may be particularly effective during bacterial infections in cooler body sites (e.g., extremities, upper respiratory tract) or in combination with therapeutic hypothermia.

• Exploiting differences between bacterial species: The variation in IF2 IDR sequences across bacterial species offers potential for developing species-selective antimicrobials.

• Combination therapy approaches: Drugs targeting IF2 condensation could potentially sensitize bacteria to traditional antibiotics by disrupting their stress adaptation mechanisms.

• Screening strategies: High-throughput screens could identify compounds that either prevent IF2 condensation or lock IF2 in the condensed state, both potentially disrupting bacterial adaptation to environmental stress.

This represents a promising direction for next-generation antibiotics targeting a previously unexplored bacterial vulnerability, particularly for addressing infections in temperature-variable environments.

What are the optimal expression conditions for producing recombinant E. coli IF2?

Based on established protocols, the following expression conditions yield optimal results for recombinant E. coli IF2 production:

• Host strain selection: E. coli C600 [pCI857] has proven effective, as it carries a plasmid with a temperature-sensitive lambda CI repressor .

• Expression vector: The INFB gene should be cloned into a heat-inducible runaway plasmid like pCP40, under the control of the strong lambda PL promoter .

• Growth medium: Rich media like LB supplemented with appropriate antibiotics for plasmid maintenance.

• Temperature protocol:

  • Grow cells at 30°C until mid-log phase (OD600 = 0.5-0.6)

  • Induce expression by temperature shift to 42°C

  • Continue expression at 37°C for 2 hours

• Harvest timing: Optimal protein yield is typically achieved 2 hours post-induction .

This protocol can achieve expression levels approximately 30 times higher than wild-type cells , making it suitable for large-scale production of IF2 for structural and functional studies.

How can one design experiments to distinguish the roles of different IF2 isoforms?

E. coli produces three IF2 isoforms with varying N-terminal regions. To distinguish their roles:

• Isoform-specific expression systems:

  • Design constructs expressing only specific isoforms

  • Create strains where endogenous IF2 is replaced with single isoforms

  • Assess growth under various conditions (temperature, nutrients)

• Complementation experiments:

  • Use IF2-depleted strains to test rescue by individual isoforms

  • Particularly test cold shock recovery (15°C) where the N-terminal IDR is critical

• Phase separation comparison:

  • Purify each isoform and compare:

    • Phase separation propensity with RNA

    • Temperature sensitivity of condensation

    • RNA binding preferences

• Domain swapping:

  • Engineer chimeric proteins with domains from different isoforms

  • Test functionality in vivo and condensation properties in vitro

• Ribosome binding and GTP hydrolysis:

  • Compare kinetic parameters of each isoform

  • Assess initiation complex formation efficiency

Data from the search results indicate that the β-isoform (lacking the first 158 amino acids) shows a robust loss in phase separation compared to the α-isoform, while still maintaining proper GTP binding . This suggests functional differences between isoforms that may be relevant to their roles under different growth conditions.

What quantitative methods are available for measuring IF2 phase separation properties?

Several quantitative methods can effectively measure IF2 phase separation:

• Turbidity measurements:

  • Measure absorbance at 350 nm after mixing IF2 with RNA

  • Track turbidity changes in response to temperature, salt, or other variables

  • Quantify the kinetics of phase separation and dissolution

• Fluorescence microscopy quantification:

  • Use fluorescently labeled IF2 or RNA

  • Measure:

    • Number of droplets per field

    • Average droplet size

    • Droplet size distribution

    • Intensity within droplets vs. surrounding solution

• Fluorescence recovery after photobleaching (FRAP):

  • Assess molecular dynamics within condensates

  • Determine exchange rates between condensed and solution phases

  • Compare mobility across temperature conditions

• Differential interference contrast (DIC) microscopy:

  • Allow label-free visualization and quantification of droplets

  • Measure changes in droplet properties under different conditions

• Partition coefficient determination:

  • Measure the concentration ratio of IF2 in condensed vs. dilute phases

  • Compare partitioning of different molecular partners

For in vivo studies, quantifying the percentage of cells showing IF2 puncta after cold shock and during recovery provides valuable information about the physiological relevance of condensation . These methods collectively provide a comprehensive assessment of phase separation behavior and its biological significance.

What are common challenges in working with recombinant IF2 and how can they be addressed?

Researchers working with recombinant E. coli IF2 frequently encounter several challenges:

• Protein solubility issues:

  • Challenge: The large IDR can lead to aggregation during expression or purification

  • Solution: Optimize buffer conditions (consider adding low concentrations of detergents or arginine), use lower induction temperatures, and employ careful purification protocols

• Proper folding verification:

  • Challenge: Ensuring the recombinant protein is correctly folded

  • Solution: Verify activity through mant-GTP binding assays , GTPase activity tests, and circular dichroism

• Preventing premature condensation:

  • Challenge: IF2 may undergo unwanted phase separation during handling

  • Solution: Maintain high salt concentrations (>300 mM KCl) in purification buffers , avoid low temperatures during purification if studying phase separation

• Isoform heterogeneity:

  • Challenge: E. coli naturally produces multiple IF2 isoforms

  • Solution: Use constructs with defined start sites, confirm by western blot that the desired isoform is the major product

• RNase contamination:

  • Challenge: RNase contamination can interfere with RNA-dependent phase separation studies

  • Solution: Include RNase inhibitors, use RNase-free reagents, and test buffers for RNase activity

By addressing these challenges proactively, researchers can significantly improve the quality and reliability of their IF2 experiments.

How should experimental conditions be optimized for studying temperature-dependent IF2 condensation?

To effectively study temperature-dependent IF2 condensation, several experimental parameters require careful optimization:

• Buffer composition:

  • Physiologically relevant buffers (e.g., HEPES pH 7.5)

  • Controlled salt concentration (50-150 mM KCl for phase separation studies)

  • Presence/absence of nucleotides (GTP/GDP) based on experimental goals

• Temperature control:

  • Precise temperature regulation for both cold shock (4°C) and recovery (37°C)

  • Gradual temperature transitions to mimic natural environmental changes

  • Consistent timing (e.g., 1 hour cold shock, 1 hour recovery)

• RNA considerations:

  • Source of RNA (total cellular RNA vs. specific RNA species)

  • RNA:protein ratio optimization (typically 0.5-2:1 by mass)

  • RNA integrity verification before experiments

• Imaging parameters:

  • For in vitro droplets: appropriate surface treatments to prevent non-specific adhesion

  • For in vivo studies: fixation protocols that preserve condensates (4% formaldehyde, 4°C, 4 hours for cold-shocked samples)

• Controls:

  • Include IF2ΔNTD to confirm IDR-dependence of observed condensation

  • Test RNase and high salt treatments to verify RNA-dependence and electrostatic nature of interactions

These optimized conditions enable reliable and reproducible studies of IF2's temperature-dependent condensation behavior, facilitating comparisons across experimental conditions and bacterial species.

What considerations are important when designing IF2 truncation constructs for structure-function studies?

When designing IF2 truncation constructs for structure-function studies, researchers should consider:

• Natural isoform boundaries:

  • E. coli naturally produces multiple IF2 isoforms (α, β, γ)

  • The β-isoform lacks the first 158 amino acids, while the γ-isoform has an even shorter N-terminus

  • Using these natural boundaries can ensure physiological relevance

• Domain preservation:

  • Maintain intact functional domains (GTP-binding, tRNA-binding)

  • Verify domain boundaries using structural predictions from AlphaFold

  • Avoid truncations that might disrupt folding of globular domains

• IDR analysis:

  • Use disorder prediction algorithms (e.g., AIUPred) to identify IDR boundaries

  • Consider creating systematic truncations within the IDR to map functional regions

  • Pay attention to charge distribution and sequence composition within the IDR

• Expression optimization:

  • Include purification tags that don't interfere with function

  • Consider codon optimization for the expression host

  • Test expression levels and solubility of each construct

• Functional verification:

  • Confirm proper folding through GTP binding assays

  • Assess activity in reconstituted translation initiation assays

  • Test complementation of IF2-depleted strains

The search results show that IF2ΔNTD (lacking the first 294 amino acids) and the β-isoform (lacking the first 158 amino acids) both fold properly as evidenced by mant-GTP binding, yet show reduced phase separation . This approach effectively connected specific regions to phase separation function while maintaining core translation activities.