Recombinant Escherichia coli O17:K52:H18 Translation initiation factor IF-2 (infB), partial

What is the primary role of Translation initiation factor IF-2 in bacterial protein synthesis?

Translation initiation factor IF-2 is one of three essential initiation factors in Escherichia coli (along with IF1 and IF3) that facilitates efficient and accurate de novo translation initiation. The primary function of IF-2 is to facilitate the binding of initiator formylmethionyl-tRNA (fMet-tRNA^fMet) to the P-site of the 30S initiation complex (IC). This interaction is crucial for the correct positioning of the initiator tRNA at the start codon of the mRNA during the formation of the 30S initiation complex . IF-2 also plays a critical role in controlling translation fidelity by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes that carry an N-formyl-methionyl-tRNA .

How does the structure of IF-2 relate to its functional properties?

IF-2 has a multi-domain structure where each domain contributes to specific aspects of its function. Research has identified that domain III of IF-2 plays a pivotal, allosteric role in IF-2 activation, suggesting it functions as a regulatory element in translation initiation. This domain undergoes a GTP- and fMet-tRNA^fMet-dependent conformational switch that is necessary for rapid 50S subunit joining . The structural elements of IF-2 are arranged to allow for interaction with both the ribosome and the initiator tRNA simultaneously, which enables it to position the initiator tRNA correctly at the P-site. This conformational flexibility is essential for IF-2's role in translation initiation and represents a potential target for antimicrobial drug development .

Why is the infB gene considered essential in bacterial systems?

The infB gene, which encodes IF-2, is essential for bacterial viability because it produces a protein required for efficient translation initiation. Experimental evidence confirms this essentiality through knockout studies where the infB gene was replaced with a kanamycin resistance marker (ΔinfB::Kan^R). In these experiments, viable transductants were only obtained when a plasmid-borne copy of IF-2 was present to complement the chromosomal deletion . The essentiality of infB underscores the critical role of IF-2 in bacterial protein synthesis and highlights why it has been considered a potential target for antimicrobial development.

How does IF-2 coordinate with other initiation factors during translation initiation?

IF-2 functions in concert with IF1 and IF3 to form a synchronized initiation complex. While IF-2 primarily facilitates binding of fMet-tRNA^fMet to the P-site, IF3 works complementarily to enhance the fidelity of this process by destabilizing 30S initiation complexes containing non-initiator tRNAs or non-canonical codon-anticodon pairings . The three initiation factors bind to the 30S subunit and synergistically regulate the kinetics of tRNA binding, ensuring that fMet-tRNA^fMet is preferentially selected for the P-site where it base-pairs with the start codon .

Methodologically, researchers investigating these interactions typically employ a combination of biochemical assays (such as filter binding assays, toe-printing), structural studies (cryo-EM, X-ray crystallography), and fluorescence-based techniques (FRET) to monitor the assembly of initiation complexes and the conformational changes in the components.

What is the mechanistic basis for the GTP-dependent conformational switch in IF-2?

The activation of IF-2 involves a GTP- and fMet-tRNA^fMet-dependent conformational switch that is essential for its function in promoting 50S subunit joining. Single-molecule fluorescence resonance energy transfer (FRET) studies have directly observed this conformational switch within 30S initiation complexes that lack IF3 .

The mechanistic model suggests that:

• GTP binding induces initial conformational changes in IF-2

• Interaction with fMet-tRNA^fMet further stabilizes the activated conformation

• Domain III of IF-2 plays an allosteric role in regulating this conformational switch

• The activated conformation of IF-2 promotes rapid 50S subunit joining

This conformational switch represents a critical checkpoint in translation initiation that ensures only correctly formed initiation complexes proceed to the elongation phase of protein synthesis .

How does IF-2 contribute to translation re-initiation in bacterial systems?

Translation re-initiation is a process where ribosomes that have terminated translation of an upstream open reading frame (ORF) can reinitiate translation at a downstream ORF without dissociating from the mRNA. Research using dicistronic reporter systems based on the translationally coupled geneV-geneVII pair from M13 phage has demonstrated that IF-2 is required for efficient re-initiation .

The methodological approach to studying re-initiation typically involves:

• Construction of dicistronic reporters with varying intercistronic distances

• Modulation of IF-2 expression levels

• Use of mutant initiator tRNAs to assess the importance of formylation and P-site binding

• Quantification of translation efficiency of both cistrons

The results show that two unique properties of bacterial initiator tRNA—formylation and binding to the ribosomal P-site—are as important for re-initiation as they are for de novo initiation, and that IF-2 plays a crucial role in facilitating this process .

What methods are most effective for analyzing the conformational dynamics of IF-2?

To effectively analyze the conformational dynamics of IF-2, researchers typically employ a combination of techniques:

Single-molecule FRET has been particularly valuable for directly observing the GTP- and fMet-tRNA^fMet-dependent conformational switch in IF-2 within 30S initiation complexes, providing insights into how this switch is regulated .

What are the methodological approaches for studying the interaction between IF-2 and fMet-tRNA^fMet?

Studying the interaction between IF-2 and fMet-tRNA^fMet requires specialized techniques that can capture both binding affinity and functional consequences:

• Filter binding assays: Quantify the binding affinity between purified IF-2 and radiolabeled fMet-tRNA^fMet

• Surface plasmon resonance: Measure real-time binding kinetics between IF-2 and fMet-tRNA^fMet

• Chemical cross-linking followed by mass spectrometry: Identify specific interaction sites

• Fluorescently labeled components: Monitor binding events in real-time

• Ribosome binding assays: Assess the effect of mutations in either component on 30S IC formation

• Toe-printing assays: Determine the position of the ribosome on mRNA and evaluate the effect of IF-2 on initiator tRNA positioning

These methodologies have revealed that IF-2 specifically recognizes the formyl group on Met-tRNA^fMet, which is crucial for its function in translation initiation .

How can genetic complementation assays be designed to evaluate IF-2 function?

Genetic complementation assays provide powerful tools for evaluating IF-2 function in vivo. A methodological approach based on the search results would include:

• Generation of conditional IF-2 mutants:

  • Create a strain where the chromosomal infB gene is replaced with an antibiotic resistance marker (e.g., ΔinfB::Kan^R)

  • Provide wild-type IF-2 on a temperature-sensitive or inducible plasmid

• Complementation testing:

  • Transform the conditional mutant with plasmids expressing variant IF-2 proteins

  • Test for growth under non-permissive conditions

  • Quantify growth rates to assess the degree of complementation

• Domain swapping experiments:

  • Create chimeric IF-2 proteins by swapping domains between bacterial and eukaryotic homologs

  • Test for functional complementation

• Cross-species complementation:

  • Test whether IF-2 from different species (e.g., bovine mitochondrial IF-2) can functionally replace E. coli IF-2

This approach has been successfully used to demonstrate that bovine mitochondrial IF-2 can functionally replace E. coli IF-2 in transduction experiments, indicating conservation of core functions despite sequence divergence .

What expression systems are optimal for producing functional recombinant IF-2?

The choice of expression system for producing functional recombinant IF-2 is critical for downstream applications. Based on the literature and experimental considerations, the following systems have been successfully employed:

For bacterial IF-2, the E. coli BL21(DE3) system is typically preferred due to its high yield and the native environment it provides for bacterial protein folding. The addition of a His-tag at either the N- or C-terminus facilitates purification while generally preserving functional activity.

What purification strategies yield the highest activity recombinant IF-2?

Purification of recombinant IF-2 with high activity requires careful attention to protein stability and functional preservation. A methodological approach typically includes:

• Initial capture step:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged IF-2

  • Ensure buffer conditions (pH 7.5-8.0, 150-300 mM NaCl) maintain stability

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose) to separate charged variants

  • Buffer containing 5-10% glycerol and 1-2 mM DTT helps maintain activity

• Polishing step:

  • Size exclusion chromatography to remove aggregates and ensure monodispersity

  • Consider including GTP or non-hydrolyzable GTP analogs to stabilize active conformation

• Quality control assessments:

  • GTPase activity assay

  • 30S binding assay

  • fMet-tRNA^fMet binding assay

  • Thermal stability analysis

Throughout the purification process, it's essential to minimize exposure to proteases and to include protease inhibitors in early purification steps. The inclusion of glycerol (10%) and reducing agents in storage buffers helps maintain long-term stability and activity of the purified protein.

How can the GTPase activity of recombinant IF-2 be accurately measured?

Measuring the GTPase activity of recombinant IF-2 is essential for assessing its functional integrity. Several methodological approaches can be employed:

• Colorimetric phosphate detection:

  • Malachite green assay to detect released inorganic phosphate

  • Advantages: Simple, inexpensive, can be adapted to plate reader format

  • Limitations: Less sensitive, potential interference from buffer components

• Radioactive assays:

  • Using [γ-³²P]GTP and measuring release of ³²P-labeled inorganic phosphate

  • Advantages: Highly sensitive, direct measurement

  • Limitations: Requires radioisotope handling, specialized equipment

• Fluorescent or bioluminescent assays:

  • Coupled enzyme assays that link GTP hydrolysis to a fluorescent or luminescent output

  • Advantages: High sensitivity, real-time monitoring capability

  • Limitations: Potential for coupling enzyme to become rate-limiting

• Ribosome-dependent GTPase activity:

  • Measuring GTPase activity in the presence of 70S ribosomes, mRNA, and fMet-tRNA^fMet

  • Provides context-specific activity measurements that reflect the physiological function

For accurate measurements, it's crucial to include appropriate controls (e.g., a GTPase-inactive IF-2 variant) and to optimize reaction conditions (temperature, pH, Mg²⁺ concentration) to reflect physiological conditions while maximizing assay sensitivity.

How can IF-2 be utilized as a target for antibiotic development?

Research has identified domain III of IF-2 as playing a pivotal, allosteric role in its activation, suggesting this domain could be targeted for the development of novel antibiotics . A methodological approach to exploiting IF-2 as an antibiotic target would include:

• Structure-based drug design:

  • Using high-resolution structures of IF-2, particularly domain III

  • In silico screening of compound libraries against specific binding pockets

  • Design of peptidomimetics that interfere with key protein-protein interactions

• High-throughput screening approaches:

  • Development of GTPase activity assays adaptable to HTS format

  • Screening for compounds that inhibit the conformational switch measured by FRET

  • Cell-based assays measuring translation efficiency in the presence of candidate compounds

• Validation in cellular systems:

  • Testing candidate compounds for specific inhibition of bacterial translation

  • Confirming target engagement using genetic approaches (e.g., resistant mutants)

  • Evaluating spectrum of activity across different bacterial species

• Optimization pipeline:

  • Structure-activity relationship studies to improve potency and specificity

  • Medicinal chemistry optimization for pharmacokinetic properties

  • Testing in animal models of infection

The essential nature of IF-2 and its conservation across bacterial species make it an attractive antibiotic target, especially if compounds can be developed that specifically target bacterial IF-2 without affecting mammalian homologs.

What research applications benefit from using recombinant IF-2 in in vitro translation systems?

Recombinant IF-2 is a valuable component for several research applications involving in vitro translation systems:

• Reconstituted translation initiation systems:

  • Study the molecular mechanisms of translation initiation

  • Assess the effects of mutations in translation components

  • Investigate the role of specific domains in IF-2 function

• Synthetic biology applications:

  • Development of optimized cell-free protein synthesis systems

  • Engineering orthogonal translation systems with altered specificity

  • Creation of minimal translation systems for specific applications

• Structural biology studies:

  • Cryo-EM studies of translation initiation complexes

  • Single-molecule studies of ribosome assembly

  • Understanding the dynamics of translation initiation

• Drug screening platforms:

  • Assays for compounds that inhibit translation initiation

  • Evaluation of resistance mechanisms to translation inhibitors

  • Counter-screens to assess specificity of translation inhibitors

• Teaching and demonstration:

  • Educational kits demonstrating the principles of translation

  • Hands-on laboratory exercises in molecular biology courses

The availability of well-characterized recombinant IF-2 has significantly advanced our understanding of translation initiation mechanics and continues to enable new research directions in both basic and applied science.

How is IF-2 function being studied in the context of translation reinitiation research?

Translation reinitiation, where ribosomes resume translation at a downstream start codon after completing translation of an upstream open reading frame, is an important regulatory mechanism. Research on IF-2's role in reinitiation typically employs these methodological approaches:

• Dicistronic reporter systems:

  • Construction of reporter plasmids containing two consecutive open reading frames

  • Based on naturally coupled genes such as the geneV-geneVII pair from M13 phage

  • Quantification of translation efficiency of both cistrons under various conditions

• Manipulation of IF-2 levels:

  • Conditional depletion or overexpression of IF-2

  • Expression of mutant forms of IF-2 with altered function

  • Assessment of the effects on reinitiation efficiency

• Analysis of reinitiation requirements:

  • Testing the importance of initiator tRNA formylation

  • Evaluating the role of Shine-Dalgarno sequences

  • Measuring the effects of intercistronic distance

• Computational modeling:

  • Development of mathematical models of reinitiation

  • Prediction of reinitiation efficiency based on sequence features

  • Integration of experimental data with computational predictions

These studies have revealed that IF-2 is required for efficient translation reinitiation in E. coli, similar to its role in de novo initiation, and that the unique properties of bacterial initiator tRNA (formylation and P-site binding) are important for both processes .

What are common issues in recombinant IF-2 expression and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant IF-2:

When expressing the partial IF-2 protein, as mentioned in the search results for "Recombinant Escherichia coli O17:K52:H18 Translation initiation factor IF-2 (infB), partial" , additional considerations may be necessary to ensure the truncated protein retains the desired structural and functional properties.

How can researchers troubleshoot issues in IF-2 activity assays?

Activity assays for IF-2 can present several technical challenges. Here's a methodological approach to troubleshooting common issues:

• Low or no GTPase activity:

  • Check Mg²⁺ concentration (typically 5-10 mM is optimal)

  • Verify GTP quality and concentration

  • Ensure proper folding of IF-2 (native gel electrophoresis can help assess this)

  • Test activity in the presence of ribosomes and fMet-tRNA^fMet (physiological activators)

• High background in translation initiation assays:

  • Perform thorough RNase treatment of components before assay

  • Ensure high purity of all components (especially ribosomes and tRNAs)

  • Include appropriate negative controls (e.g., without mRNA or with non-cognate start codons)

  • Consider using nuclease-treated extracts for in vitro translation

• Poor reproducibility:

  • Standardize protein storage conditions to maintain activity

  • Prepare fresh dilutions of IF-2 for each experiment

  • Carefully control temperature during experiments

  • Consider batch effects in ribosome and tRNA preparations

• Issues with fMet-tRNA^fMet binding assays:

  • Verify the aminoacylation and formylation status of tRNA

  • Optimize buffer conditions (particularly pH and salt concentration)

  • Consider the native conformational state of IF-2 (GTP-bound form has higher affinity)

By systematically addressing these issues, researchers can improve the reliability and sensitivity of IF-2 activity assays, leading to more robust experimental results.

What controls are essential when studying the conformational dynamics of IF-2?

• Nucleotide state controls:

  • GTP (active state)

  • GDP (inactive state)

  • Non-hydrolyzable GTP analogs (e.g., GDPNP, GTPγS) to trap specific conformational states

  • Nucleotide-free conditions as baseline

• Protein variant controls:

  • GTPase-deficient mutants (typically mutations in the G-domain)

  • Domain III mutants that affect allosteric regulation

  • Truncated constructs lacking specific domains

• Interaction partner controls:

  • Complete 30S initiation complex

  • 30S subunits without mRNA or tRNA

  • Presence/absence of IF1 and IF3

  • Non-formylated Met-tRNA^Met to test formyl-group specificity

• Environmental controls:

  • Temperature dependence of conformational changes

  • Mg²⁺ concentration variations

  • Buffer composition effects

• Methodological controls:

  • For FRET experiments: single-labeled constructs, directly excited acceptor

  • For structural studies: sample homogeneity verification, grid quality assessment

  • For functional assays: heat-inactivated IF-2, competition with excess unlabeled components

Implementing these controls allows researchers to distinguish specific IF-2 conformational changes from artifacts and to correlate structural dynamics with functional outcomes.

What emerging technologies are advancing our understanding of IF-2 function?

Several cutting-edge technologies are transforming our ability to study IF-2 function:

• Cryo-electron microscopy advances:

  • Time-resolved cryo-EM capturing transient states during initiation

  • Improved resolution allowing visualization of side-chain conformations

  • Computational sorting of conformational ensembles

• Single-molecule techniques:

  • Multi-color FRET to simultaneously track multiple components

  • Combined force and fluorescence microscopy to correlate structure and mechanics

  • Zero-mode waveguides for studying initiation at physiological concentrations

• Mass spectrometry innovations:

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Cross-linking mass spectrometry for interaction mapping

  • Native mass spectrometry for stoichiometry and complex integrity analysis

• Computational approaches:

  • Molecular dynamics simulations with improved force fields

  • Machine learning for predicting functional effects of mutations

  • Integrative modeling combining data from multiple experimental sources

• Genome editing technologies:

  • CRISPR-based approaches for precise genomic manipulation of IF-2

  • Creation of conditional alleles for in vivo studies

  • High-throughput mutational scanning of IF-2 function

These technologies promise to provide unprecedented insights into the mechanics of IF-2 function, particularly regarding the conformational dynamics that underlie its role in translation initiation.

How might research on IF-2 contribute to our understanding of bacterial adaptation and evolution?

Research on IF-2 has significant implications for understanding bacterial adaptation and evolution:

• Evolutionary conservation and divergence:

  • Comparative analysis of IF-2 across bacterial phyla

  • Investigation of co-evolution with ribosomal components

  • Understanding how IF-2 adapts to different environmental niches

• Bacterial stress responses:

  • Role of IF-2 in translation regulation during stress conditions

  • Potential post-translational modifications affecting IF-2 function

  • Changes in IF-2 expression levels in response to environmental challenges

• Antibiotic resistance mechanisms:

  • Adaptation of translation initiation machinery in resistant strains

  • Compensatory mutations in IF-2 in response to ribosome-targeting antibiotics

  • Design of combination therapies targeting multiple translation components

• Horizontal gene transfer implications:

  • Compatibility of IF-2 with foreign mRNAs and tRNAs

  • Role in expression of horizontally acquired genes

  • Potential barriers to gene transfer related to translation initiation

• Synthetic biology applications:

  • Engineering IF-2 variants with altered specificity

  • Development of orthogonal translation systems

  • Creation of minimal cells with streamlined translation machinery

By exploring these aspects, researchers can gain insights into the fundamental processes that drive bacterial adaptation and evolution, with potential applications in fields ranging from infectious disease treatment to synthetic biology.

What are the potential applications of engineered IF-2 variants in biotechnology?

Engineered IF-2 variants hold promise for several biotechnology applications:

• Enhanced protein production systems:

  • IF-2 variants with increased initiation efficiency

  • Engineered specificity for non-canonical start codons

  • Temperature-optimized variants for cold-adapted expression systems

• Synthetic biology tools:

  • Orthogonal translation initiation systems for genetic isolation

  • Inducible initiation factors for tight regulation of gene expression

  • Components for artificial cells or minimal synthetic systems

• Therapeutic applications:

  • Target for narrow-spectrum antibiotics

  • Delivery of modified IF-2 to selectively inhibit bacterial translation

  • Diagnostic tools based on IF-2 interactions

• Research reagents:

  • Labeled IF-2 variants for structural and functional studies

  • IF-2-based affinity tags for purification of translation complexes

  • Sensors for translation initiation events in living cells

• Educational tools:

  • Simplified in vitro translation systems for teaching

  • Visualization tools for translation initiation

  • Modular components for synthetic biology education

The development of these applications requires a deep understanding of IF-2 structure-function relationships, which continues to be advanced through basic research into this essential translation factor.