Recombinant Escherichia coli O81 Translation initiation factor IF-2 (infB), partial

What is translation initiation factor IF-2 and what role does it play in bacterial protein synthesis?

Translation initiation factor 2 (IF-2) is a GTPase protein essential for the initiation of protein synthesis in bacteria. It performs several critical functions during the translation initiation process. The primary role of IF-2 is to ensure the fidelity of translation initiation by selectively promoting the joining of the 50S ribosomal subunit to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNA^fMet) . This selective function ensures that translation begins with the correct initiator tRNA at the correct start codon. Additionally, IF-2 helps position the initiator tRNA in the P-site of the ribosome and interacts with GTP, with hydrolysis occurring after 50S subunit joining. The conformational dynamics of IF-2 serve as a molecular checkpoint in the initiation pathway, verifying the presence of the correct initiator tRNA before allowing progression to the elongation phase of protein synthesis .

How does the infB gene organize in bacterial genomes and how is its expression regulated?

The infB gene, which encodes translation initiation factor IF-2, is typically organized within operons in bacterial genomes. In Bacillus subtilis, for example, the infB gene is part of the nusA-infB operon . The interval between nusA and infB contains two short open reading frames potentially encoding polypeptides of 10,402 and 11,046 Mr . This operon organization suggests coordinated expression of these genes involved in transcription and translation processes. The gene organization can be visualized as shown in Figure 1 of the source material, where open reading frames are represented as open boxes with transcriptional initiation and termination signals indicated . Primer extension analysis and other molecular techniques have been used to identify the promoter regions controlling expression of this operon, with experiments involving conditional disruption of the infB operon confirming the location of the promoter in specific genomic regions .

What is the molecular weight and domain structure of E. coli IF-2?

Escherichia coli translation initiation factor IF-2 is a protein with a molecular mass of approximately 97 kDa . Structure-function studies have led to the proposal of a six-domain structural model for IF-2 . According to this model, IF-2 is a slightly elongated molecule with:

• A structurally compact C-terminal domain that interacts with the ribosome and fMet-tRNA^fMet

• A well-conserved central GTP-binding domain responsible for GTPase activity

• A highly charged, solvent-exposed N-terminal region with protruding α-helical structures that interacts with the 30S subunit

What techniques are most effective for studying the conformational dynamics of IF-2?

Understanding the conformational changes of IF-2 during translation initiation requires sophisticated experimental approaches. Single-molecule fluorescence resonance energy transfer (smFRET) has proven particularly valuable and has directly observed the conformational switch associated with IF-2 activation within 30S initiation complexes . This technique involves labeling IF-2 and tRNA with fluorescent dyes and measuring energy transfer between them as a function of their distance, providing real-time information about conformational changes in individual molecules.

Other complementary techniques include:

The GTP- and fMet-tRNA^fMet-dependent "activation" of IF-2 involves a conformational switch that can be directly monitored using these techniques . Domain III of IF-2 plays a pivotal, allosteric role in controlling this conformational switch, suggesting that structural signals are transmitted between different domains of the protein to coordinate its activity .

How can researchers map epitopes and structural features of IF-2?

Researchers employ multiple complementary approaches to map epitopes and structural features of IF-2. One effective strategy involves raising and characterizing monoclonal antibodies against E. coli IF-2, which allows for the identification of surface epitopes on the protein . In combination with this approach, researchers create a series of nested deletion mutants of IF-2 and test their interaction with monoclonal antibodies to localize specific epitopes to regions of the protein .

Competition immunoassays provide additional insights by determining the relative locations of different epitopes through analysis of whether different antibodies compete for binding to IF-2 . Cross-reactivity analysis, where antibodies raised against E. coli IF-2 are tested for reactivity with IF-2 from other bacterial species (such as Salmonella typhimurium, Klebsiella oxytoca, Enterobacter cloacae, Proteus vulgaris, and Bacillus stearothermophilus), helps identify conserved epitopes that may be functionally important .

These experimental approaches can be enhanced by computational predictions of secondary structure, which help researchers understand the folding of the protein and interpret experimental results in the context of the six-domain structural model of IF-2 . The integration of these methods provides a comprehensive picture of IF-2's structure and surface features, which informs models of how the protein functions in translation initiation.

What expression and purification strategies are optimal for obtaining functional recombinant IF-2?

Expressing and purifying recombinant IF-2 for research requires careful consideration of several factors to maintain functionality. E. coli expression systems using vectors with inducible promoters (T7 or tac) are commonly employed, often with fusion tags (His, GST, or MBP) to facilitate purification and enhance solubility. Expression conditions must be optimized, with lower temperatures (16-25°C) generally improving the solubility of large proteins like IF-2.

A typical purification strategy might include:

• Affinity chromatography (e.g., Ni-NTA for His-tagged IF-2)

• Ion exchange chromatography (exploiting IF-2's charge distribution)

• Size exclusion chromatography (final polishing step)

• Optional heparin affinity chromatography (helpful due to IF-2's nucleic acid binding properties)

Quality control is essential, with SDS-PAGE, Western blotting, mass spectrometry, and functional assays (GTP binding/hydrolysis, tRNA binding) confirming identity, purity, and activity. For storage, IF-2 is typically kept in buffers containing 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0), 100-200 mM KCl or NaCl, 1-10 mM MgCl₂, 1-5 mM DTT, and 10-20% glycerol, then flash-frozen in liquid nitrogen and stored at -80°C to preserve activity.

Researchers must carefully validate the functional properties of purified IF-2 to ensure that it retains native conformational dynamics and activities, particularly for studies focused on the conformational switch that controls translation initiation fidelity .

How does IF-2 ensure the fidelity of translation initiation?

IF-2 ensures the fidelity of translation initiation through a sophisticated molecular mechanism centered on its ability to selectively accelerate 50S ribosomal subunit joining. This selectivity is achieved through several coordinated processes:

• Selective acceleration: IF-2 selectively increases the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNA^fMet) . This preferential acceleration for correctly formed initiation complexes serves as a quality control mechanism.

• Conformational activation: A GTP- and fMet-tRNA^fMet-dependent "activation" of IF-2 involves a conformational switch that has been directly observed using single-molecule fluorescence resonance energy transfer (FRET) . This activation occurs only when the correct initiator tRNA is present in the 30S IC.

• Allosteric regulation: Domain III of IF-2 plays a pivotal, allosteric role in controlling this conformational switch , suggesting that structural signals are transmitted between different domains of the protein to coordinate its activity.

• GTPase activity: The GTP-binding and hydrolysis activities of IF-2 are integrated with its conformational changes to couple energy expenditure with the verification of correct initiator tRNA selection.

Through these mechanisms, IF-2 effectively discriminates between initiation complexes containing the correct initiator tRNA (fMet-tRNA^fMet) and those containing incorrect tRNAs, thereby preventing translation from beginning at inappropriate sites or with incorrect amino acids .

What is the relationship between IF-2's GTPase activity and its role in translation initiation?

The GTPase activity of IF-2 is intricately linked to its role in translation initiation through a cycle of conformational changes and molecular interactions:

• GTP binding induces activation: When GTP binds to IF-2, it promotes a conformational change that activates the protein, enabling it to efficiently interact with the initiator tRNA and the ribosome .

• Conformational signaling: The GTP-bound conformation of IF-2 signals the readiness of the 30S initiation complex for 50S subunit joining, but only when the correct fMet-tRNA^fMet is present .

• GTP hydrolysis timing: GTP hydrolysis occurs after 50S subunit joining, converting IF-2 from its GTP-bound "active" conformation to a GDP-bound "inactive" conformation.

• Factor release: The conformational change following GTP hydrolysis reduces IF-2's affinity for the ribosome, facilitating its release and allowing translation elongation to proceed.

The well-conserved central GTP-binding domain of IF-2, as identified in structural studies , contains the machinery necessary for this GTPase cycle. The regulation of IF-2's GTPase activity is a key control point in the translation initiation pathway, ensuring both fidelity and efficiency of the process by coupling nucleotide hydrolysis with conformational changes that verify correct initiator tRNA selection .

How does IF-2 interact with other translation initiation factors and the ribosome?

IF-2 functions within a complex network of interactions involving other initiation factors, the ribosome, and translation substrates. The initiation of bacterial protein synthesis involves the assembly of a 30S initiation complex (IC) composed of the small (30S) ribosomal subunit, initiation factor (IF) 1, IF-2, IF-3, initiator fMet-tRNA^fMet, and messenger RNA (mRNA) . These components work synergistically to ensure accurate start codon selection and correct positioning of the initiator tRNA.

IF-1, IF-2, and IF-3 bind to the 30S subunit and synergistically regulate the kinetics of tRNA binding . This coordinated action helps ensure that fMet-tRNA^fMet is preferentially selected into the peptidyl-tRNA-binding (P) site of the 30S subunit, where it base-pairs to the start codon of an mRNA . IF-3 plays a role in verifying the authenticity of the start codon and initiator tRNA, while IF-1 influences the positioning of the initiator tRNA and affects IF-2 function.

The six-domain structural model of IF-2 provides a framework for understanding these interactions . The N-terminal domain, characterized by its charged, solvent-exposed nature with protruding α-helical structures, interacts with the 30S ribosomal subunit . The central GTP-binding domain interacts with GTP and coordinates the energy-dependent steps of initiation. The C-terminal domain, which is structurally compact, is primarily responsible for recognition of and interaction with the initiator tRNA .

How does the conformational switch in IF-2 relate to antibiotic development opportunities?

The conformational switch in IF-2 presents promising opportunities for antibiotic development, particularly given the rising challenge of antimicrobial resistance. Research has identified that domain III of IF-2 plays a pivotal, allosteric role in the protein's activation . This domain can potentially be targeted for the development of novel antibiotics , with several strategic advantages:

• Targeting allosteric sites rather than active sites may present unique selectivity advantages

• The essential nature of IF-2 for bacterial protein synthesis makes it a valuable target

• Structural differences between bacterial IF-2 and eukaryotic eIF5B may allow selective targeting

• The conserved nature of IF-2 across bacterial species suggests broad-spectrum potential

Potential approaches for IF-2-targeted antibiotic development include:

The detailed understanding of IF-2's conformational dynamics provided by techniques such as single-molecule FRET creates a foundation for developing novel antibiotics that could address antimicrobial resistance by targeting a fundamental bacterial process through a mechanism distinct from existing antibiotics.

What are the species-specific variations in IF-2 structure and function?

Variations in the infB gene across bacterial species create important functional differences that impact both basic understanding of translation mechanisms and approaches to antibiotic development. Structural studies and comparative analyses reveal patterns of conservation and divergence:

• The central GTP-binding domain is highly conserved across bacterial species , reflecting its essential role in IF-2 function

• N-terminal regions show greater variability between species , suggesting adaptation to species-specific requirements

• Cross-reactivity analyses of monoclonal antibodies against IF-2 from different species (including Salmonella typhimurium, Klebsiella oxytoca, Enterobacter cloacae, Proteus vulgaris, and Bacillus stearothermophilus) reveal distinct patterns of epitope conservation

The genetic organization of infB also varies between species. In Bacillus subtilis, the infB gene is part of the nusA-infB operon, with the interval between nusA and infB containing two short open reading frames potentially encoding polypeptides of 10,402 and 11,046 Mr . This differs from the arrangement in other bacterial species, suggesting divergent evolutionary paths for translational regulation.

These variations have important implications for both fundamental research and applied studies:

• Conserved regions present opportunities for broad-spectrum antibiotics

• Variable regions may enable species-selective targeting

• Structural analysis of IF-2 across species can identify optimal targeting strategies

• Species-specific functional variations may influence susceptibility to IF-2 inhibitors

Understanding these variations is crucial for developing a complete picture of bacterial translation mechanisms and for designing effective targeted therapeutics.

How can computational approaches enhance our understanding of IF-2 conformational dynamics?

Advanced computational approaches offer powerful tools for understanding IF-2 conformational dynamics and predicting how mutations might affect function. These methods can complement experimental techniques like single-molecule FRET that have directly observed the conformational switch in IF-2 :

• Molecular dynamics (MD) simulations can model the conformational flexibility of IF-2 at different time scales, revealing transition pathways between the conformational states observed in experimental studies

• Structure prediction methods like AlphaFold2 and RoseTTAFold can predict structures of IF-2 domains or complexes where experimental structural data is lacking

• Network analysis can identify allosteric pathways connecting the GTP-binding site, domain III (with its pivotal allosteric role) , and other functional regions of the protein

• Docking algorithms can predict binding modes of IF-2 with tRNA, GTP, and ribosomal components, providing insights into recognition mechanisms

These computational approaches are particularly valuable for studying a protein like IF-2 that undergoes complex conformational changes, as they provide detailed atomic-level insights that are difficult to obtain experimentally. The integration of computational predictions with experimental data, such as using FRET measurements as constraints in simulations , enhances the biological relevance of the models and leads to more accurate mechanistic understandings of IF-2 function.

The insights gained from computational studies can guide the design of experiments to test specific hypotheses about IF-2 conformational dynamics and function, and can inform the development of small molecules targeting the conformational switch for antibiotic development.

What factors influence the conformational switch mechanism in IF-2?

The conformational switch in IF-2 is regulated by multiple factors that collectively ensure proper timing and specificity in translation initiation. Understanding these factors is essential for both mechanistic insights and therapeutic development:

• GTP binding: The binding of GTP to the well-conserved central GTP-binding domain of IF-2 triggers conformational changes that activate the protein.

• fMet-tRNA^fMet interaction: The presence of initiator tRNA (fMet-tRNA^fMet) contributes to the activation of IF-2, promoting the conformational change that has been directly observed using single-molecule FRET .

• Domain III allosteric effects: Domain III of IF-2 plays a pivotal, allosteric role in controlling the conformational switch . This domain appears to transmit structural signals between other domains of the protein to coordinate activity.

• Ribosomal interactions: Binding to the 30S ribosomal subunit likely influences the conformational landscape of IF-2, potentially priming it for activation.

• Temperature and ionic conditions: These environmental factors can affect protein dynamics and the stability of different conformational states.

The activated conformation of IF-2 allows it to efficiently promote 50S ribosomal subunit joining to 30S initiation complexes carrying fMet-tRNA^fMet . This selective acceleration of subunit joining for correctly formed initiation complexes is a key mechanism by which IF-2 ensures the fidelity of translation initiation.

The complexity of this regulatory system, with multiple inputs controlling the conformational switch, provides robustness to the translation initiation process but also presents multiple potential targets for intervention, whether for basic research manipulation or therapeutic development.

How do the functions of IF-2 compare across different domains of life?

Translation initiation factor 2 is present across all domains of life, but with significant structural and functional differences that reflect the divergent evolution of translation systems:

• Bacterial IF-2 vs. Archaeal/Eukaryotic eIF5B:

  • Both are GTPases involved in translation initiation

  • Bacterial IF-2 is typically larger (97 kDa in E. coli) compared to eIF5B

  • The N-terminal domains show the greatest divergence, while the GTP-binding domains are more conserved

  • Both facilitate initiator tRNA binding, but through different mechanisms

• Domain organization differences:

  • Bacterial IF-2 has a six-domain structure as described in E. coli studies

  • Eukaryotic eIF5B has a different domain arrangement

  • The C-terminal domains responsible for tRNA binding show structural similarities despite sequence divergence

• Functional context differences:

  • Bacterial systems use N-formyl-methionyl-tRNA (fMet-tRNA^fMet) as initiator tRNA

  • Eukaryotic systems use methionyl-tRNA without formylation

  • Eukaryotic initiation involves many more factors and is cap-dependent

• Mechanistic conservation:

  • The GTPase cycle is conserved across domains

  • The conformational switch mechanism may be a universal feature, though with domain-specific adaptations

  • The role in subunit joining appears conserved across all domains

The differences between bacterial IF-2 and its archaeal/eukaryotic counterparts provide both evolutionary insights and opportunities for selective targeting in antibiotic development. Understanding these comparative aspects helps clarify which features of the conformational switch mechanism identified in bacterial IF-2 might represent universal principles of translation initiation across all domains of life.

What are the key considerations in designing experiments to study IF-2's role in translation initiation?

Designing rigorous experiments to investigate IF-2's role in translation initiation requires careful consideration of several factors:

• System selection: Researchers must choose between in vitro reconstituted systems (providing precise control over components), cell-free translation systems (offering a more physiological environment while maintaining experimental accessibility), or in vivo approaches (providing physiological relevance but with less experimental control).

• Component preparation quality:

  • Ribosomes must be highly purified and functionally active

  • mRNAs should contain well-defined Shine-Dalgarno sequences and start codons

  • tRNAs must be correctly aminoacylated and, for initiator tRNA, properly formylated

  • IF-2 variants (wild-type, mutants, labeled) must be functionally validated

• Experimental condition optimization:

  • Buffer composition (ions, pH, crowding agents) significantly affects translation

  • Temperature controls reaction rates and complex stability

  • GTP concentration and regeneration systems maintain consistent energy supply

• Control experiments:

  • IF-2 variants with mutations in key domains can isolate specific functions

  • Comparison with other bacterial species' IF-2 can reveal conserved mechanisms

  • Negative controls without IF-2 establish baseline activities

For studies specifically focusing on the conformational switch in IF-2, single-molecule fluorescence resonance energy transfer (smFRET) has proven particularly valuable . This technique requires careful design of fluorophore labeling strategies to monitor the conformational changes associated with IF-2 activation within 30S initiation complexes .

The pivotal role of domain III in the allosteric regulation of IF-2 activation suggests that experiments targeting this domain (through mutations, deletions, or specific labeling) would be particularly informative for understanding the conformational switch mechanism.

How can researchers effectively study the interaction between IF-2 and initiator tRNA?

The interaction between IF-2 and initiator tRNA (fMet-tRNA^fMet) is central to the function of IF-2 in ensuring translation fidelity . Studying this interaction effectively requires specialized techniques:

• Binding assays:

  • Filter binding assays measure the affinity of IF-2 for fMet-tRNA^fMet

  • Surface plasmon resonance provides real-time binding kinetics

  • Microscale thermophoresis detects binding in solution with minimal sample consumption

  • These techniques can determine how GTP/GDP states affect binding affinity

• Structural approaches:

  • Cryo-electron microscopy of IF-2- fMet-tRNA^fMet complexes

  • Chemical crosslinking followed by mass spectrometry to identify contact points

  • NMR studies of specific domain interactions with tRNA

  • These methods provide spatial information about the binding interface

• Functional assays:

  • GTPase activation assays monitor how tRNA binding affects IF-2 activity

  • 50S subunit joining kinetics assays measure the selective acceleration by IF-2

  • These approaches connect physical binding to functional outcomes

• Conformational studies:

  • Single-molecule FRET directly observes the conformational switch in IF-2 associated with fMet-tRNA^fMet binding

  • Time-resolved measurements capture the dynamics of the interaction

  • These techniques reveal how tRNA binding triggers allosteric changes in IF-2

• Mutagenesis approaches:

  • Mutations in the C-terminal domain of IF-2 that interacts with tRNA

  • Modifications to the allosteric domain III that regulates the conformational switch

  • tRNA modifications that alter recognition elements

  • These strategies help identify specific residues critical for the interaction

Understanding the IF-2- fMet-tRNA^fMet interaction is essential for deciphering the mechanism by which IF-2 ensures that translation begins with the correct initiator tRNA at the correct start codon, a fundamental aspect of translation fidelity.

What techniques can identify potential antibiotic compounds targeting the IF-2 conformational switch?

The conformational switch in IF-2, particularly the allosteric role of domain III , presents opportunities for novel antibiotic development. Several techniques can identify compounds targeting this mechanism:

For compounds identified through these screens, validation studies are essential:

• Binding verification using biophysical techniques (isothermal titration calorimetry, surface plasmon resonance)

• Mechanism confirmation through structural studies (X-ray, cryo-EM) of IF-2-compound complexes

• Specificity testing against other GTPases and eukaryotic translation factors

• Cellular activity assessment in diverse bacterial species

• Resistance development studies to understand potential escape mechanisms

The ideal compound would stabilize the inactive conformation of IF-2, preventing the GTP- and fMet-tRNA^fMet-dependent activation required for efficient 50S subunit joining . By targeting the allosteric mechanism rather than the GTP-binding site directly, such compounds might achieve selectivity over other GTPases while maintaining activity against a broad spectrum of bacterial species.