Recombinant Escherichia coli O7:K1 Translation initiation factor IF-2 (infB), partial

What is the primary function of E. coli Translation Initiation Factor IF-2?

Translation Initiation Factor 2 controls the fidelity of translation initiation in bacteria by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNA fMet). This process involves a GTP- and fMet-tRNA fMet-dependent "activation" of IF2 that facilitates rapid subunit joining . IF2 also positions ribosomal subunits in a distinct rotational orientation during the subunit-joining step of initiation and stabilizes the mobile L1 stalk of the large subunit in a unique conformation .

How is the infB gene organized in E. coli?

The infB gene in E. coli spans approximately 2,670 base pairs encoding an 890 amino acid protein . It is part of a larger operon structure, and its expression is regulated at both transcriptional and post-transcriptional levels. Temperature shifts (such as from 37°C to 10°C) affect infB transcription, mRNA stability, and translation . The gene corresponds to the region between positions 3313176 and 3316155 (reverse strand) of the genomic sequence in E. coli K-12 substr. MG1655 .

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

E. coli IF2 contains several functional domains:

• N-terminal domain (variable between species but highly conserved within E. coli)

• Central GTP/GDP-binding domain (domain IV)

• C-terminal domain (essential for function)

Studies have shown that IF2-derived proteins of molecular weight 55 kDa or higher, as long as they contained the C-terminal half, supported growth of E. coli and fulfilled all known functions of IF2. Cells expressing IF2 with the C-terminal quarter of amino acids deleted were not viable . The N-terminal domains (I, II, and III) are completely conserved within E. coli strains, indicating a specific function of this region .

How does IF2 from different bacterial species compare in terms of conservation?

IF2 shows remarkable conservation patterns:

In a study of 10 clinical E. coli isolates, only one polymorphic position (Gln/Gly490) was found in the entire 890 amino acid sequence, located within the central GTP/GDP-binding domain IV of IF2 . This extreme conservation within E. coli suggests that IF2 has reached a highly defined level of structural and functional development.

What methodologies are recommended for purifying recombinant E. coli IF2?

For efficient purification of recombinant E. coli IF2, researchers have developed several approaches:

• Size-based differentiation method:

  • Create a strain expressing a shorter form of IF2 (55 kDa) in the absence of wild-type IF2

  • Express your recombinant mutant IF2 (97.3 or 79.7 kDa)

  • Purify based on the molecular weight difference between the endogenous and recombinant forms

• Specialized purification procedure:

  • A new procedure has been developed specifically for purification of large amounts of IF2a and IF2b isoforms

  • This approach allows for studying the activity of mutant IF2 proteins in vitro without contamination from wild-type initiation factor

• Immunoaffinity approach:

  • Utilize monoclonal antibodies against IF2 for selective purification

  • This approach leverages the specificity of antibody-antigen interactions to isolate IF2 from complex mixtures

The choice of method depends on the specific experimental needs and the nature of your downstream applications.

How can researchers study the conformational changes in IF2 during translation initiation?

Single-molecule Fluorescence Resonance Energy Transfer (FRET) has proven to be a powerful tool for studying the conformational dynamics of IF2 during translation initiation:

• IF2-tRNA FRET signal approach:

  • This technique directly observes the conformational switch associated with IF2 activation within 30S ICs

  • The method reveals how GTP, fMet-tRNA fMet, and specific structural elements of IF2 drive and regulate this conformational switch

  • Domain III of IF2 plays a pivotal, allosteric role in IF2 activation

• Ribosomal subunit rotation measurements:

  • FRET can be used to monitor how IF2 positions ribosomal subunits in distinct rotational orientations

  • This technique has revealed that IF2 stabilizes the ribosome in a semi-rotated conformation during the subunit-joining step of initiation

These approaches provide insights into the molecular mechanisms of IF2 function and can be used to study the effects of mutations in different domains of IF2.

What strategies can be used to create and analyze mutations in the GTP-binding site of IF2?

Several approaches have been developed for creating and analyzing mutations in the GTP-binding site of IF2:

• Site-directed mutagenesis:

  • Target the conserved sequence near the center of IF2 that is involved in GTP binding

  • Mutations in this binding site can be constructed using PCR-based mutagenesis approaches

• Complementation system for studying mutants:

  • Use a strain that survives with a short form of IF2 (55 kDa) in the absence of wild-type IF2

  • This system allows cloning and purification of specific IF2 mutants based on molecular weight difference (97.3 and 79.7 kDa)

  • The activity of the mutants can then be studied in vitro without interference from wild-type IF2

• Functional analysis methods:

  • In vitro translation assays to assess the impact of mutations on translation efficiency

  • GTP hydrolysis assays to determine changes in GTPase activity

  • Ribosome binding assays to evaluate interactions with ribosomal subunits

These tools provide a comprehensive approach for molecular studies of the structure and activities of different IF2 domains and the role of GTP in the initiation of protein biosynthesis.

How does the serotype K1 in E. coli affect IF2 function or expression?

While the K1 capsular antigen is a significant virulence factor in E. coli, particularly in strains causing neonatal meningitis and other extraintestinal infections, its specific impact on IF2 function requires careful consideration:

• Gene expression in K1 strains:

  • E. coli O1:K1:H7/NM strains, which are frequently implicated in neonatal meningitis, urinary tract infections, and septicemia, show phylogenetic differences that may affect gene expression patterns

  • The K1 capsule genes were found to be significantly more prevalent in early-onset sepsis (EOS) collections compared to other E. coli collections

• Potential research approaches:

  • Comparative transcriptomic analysis of infB expression between K1 and non-K1 strains

  • Examination of translation efficiency in the presence of the K1 capsule

  • Assessment of structural interactions between the K1 capsule and translation machinery

• Phylogenetic considerations:

  • K1 strains often belong to phylogroup B2, which was more prevalent among avian pathogenic E. coli (APEC) than among human extraintestinal pathogenic E. coli (ExPEC) (95% vs. 53%)

  • These phylogenetic differences may influence the regulatory networks controlling infB expression

Research on the specific relationship between the K1 serotype and IF2 function is an area that warrants further investigation, particularly in the context of pathogenic E. coli strains.

What are the optimal conditions for expressing recombinant E. coli IF2 in bacterial systems?

Optimizing expression of recombinant E. coli IF2 requires careful consideration of several factors:

• Host strain selection:

  • E. coli DH5α has been successfully used for transformation with recombinant plasmids containing infB

  • For studying IF2 mutants, consider using strains that allow detection of the recombinant protein against the background of endogenous IF2

• Vector systems:

  • pTZ18R has been used successfully for cloning the entire infB coding region

  • pKK232-8 with a promoterless cat gene can be used for transcriptional fusion studies

  • Plasmid selection should consider the size of the infB insert (~2.9 kbp for the entire coding region)

• Expression conditions:

  • Temperature affects infB transcription, mRNA stability, and translation

  • Standard growth is typically at 37°C in LB medium to A600 = 1

  • Cold shock conditions (10°C for 90 minutes) can be used to study temperature-dependent regulation

• Induction parameters:

  • Depending on the promoter system used, optimize induction timing and inducer concentration

  • For temperature-sensitive expression systems, determine the optimal temperature shift protocol

Monitoring expression levels through Western blotting with polyclonal anti-IF2 antibodies is recommended to confirm successful production of the recombinant protein .

How can researchers effectively study the interaction between IF2 and other translation initiation factors?

Studying the interactions between IF2 and other translation initiation factors requires specialized approaches:

• In vitro reconstitution systems:

  • Purify individual components (IF1, IF2, IF3, 30S subunits, 50S subunits, mRNA, fMet-tRNA)

  • Assemble initiation complexes in controlled conditions to study factor interactions

  • Use purified components to avoid interference from cellular factors

• Binding assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics between IF2 and other factors

  • Fluorescence anisotropy to detect changes in rotational diffusion upon complex formation

  • Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters of binding

• Structural approaches:

  • Cryo-electron microscopy to visualize IF2 within the translation initiation complex

  • Cross-linking followed by mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon binding

• Functional assays:

  • GTP hydrolysis assays to monitor the effect of factor interactions on IF2 activity

  • 30S IC formation assays to assess the cooperative effects of initiation factors

  • 50S joining assays to evaluate the impact of factor interactions on subunit association

These methods provide complementary information about the complex interplay between IF2 and other components of the translation initiation machinery.

What approaches can be used to investigate the role of IF2 in pathogenic E. coli strains compared to non-pathogenic strains?

Comparative analysis of IF2 in pathogenic versus non-pathogenic E. coli requires multi-faceted approaches:

• Genomic analysis:

  • Sequence the infB gene from multiple pathogenic (e.g., O1:K1:H7/NM) and non-pathogenic strains

  • Determine if there are consistent variations in the infB sequence or regulatory regions

  • Analyze the genomic context of infB to identify potential differences in operon structure

• Expression studies:

  • Compare infB transcription levels using qRT-PCR or RNA-seq

  • Analyze IF2 protein levels through Western blotting with anti-IF2 antibodies

  • Investigate differences in translation efficiency of infB mRNA

• Functional comparisons:

  • Create recombinant strains with swapped infB genes to assess functional differences

  • Perform in vitro translation assays using extracts from pathogenic and non-pathogenic strains

  • Evaluate the impact of IF2 variants on translation fidelity and efficiency

• Virulence correlation:

  • Create infB mutants in pathogenic strains and assess changes in virulence

  • Investigate whether IF2 interacts differently with other cellular components in pathogenic strains

  • Determine if stress conditions affect IF2 function differently in pathogenic versus non-pathogenic strains

Understanding these differences could provide insights into the potential role of IF2 in bacterial pathogenesis and adaptation.

How can researchers overcome challenges in expressing and purifying full-length IF2?

Full-length IF2 expression and purification presents several challenges that can be addressed with the following strategies:

• Expression challenges:

  • Use E. coli strains optimized for large protein expression (e.g., BL21(DE3))

  • Lower induction temperature (16-20°C) to improve folding of large proteins

  • Consider codon optimization if rare codons are present in the infB sequence

  • Express as fusion protein with solubility tags (e.g., MBP, SUMO) if solubility is an issue

• Purification strategies:

  • Implement a multi-step purification protocol:

    • Initial capture using affinity chromatography (if tagged)

    • Intermediate purification using ion exchange chromatography

    • Polishing step using size exclusion chromatography

  • Use non-ionic detergents at low concentrations if aggregation occurs

  • Include GTP or non-hydrolyzable GTP analogs in buffers to stabilize the protein

• Protein degradation prevention:

  • Include protease inhibitors in all buffers

  • Maintain samples at 4°C throughout purification

  • Consider using protease-deficient expression strains

  • Minimize freeze-thaw cycles by aliquoting purified protein

• Quality control:

  • Verify protein integrity by SDS-PAGE

  • Confirm identity by Western blotting with anti-IF2 antibodies

  • Assess activity through GTP hydrolysis assays

  • Evaluate homogeneity by dynamic light scattering

These approaches have been successfully applied to obtain purified IF2 for structural and functional studies.

What methods can be used to verify the functional activity of purified recombinant IF2?

Verifying the functional activity of purified recombinant IF2 is crucial for ensuring the validity of subsequent experiments:

• GTP hydrolysis assays:

  • Monitor the conversion of GTP to GDP by IF2 using:

    • Colorimetric assays for phosphate release

    • HPLC analysis of nucleotides

    • Radioactive GTP hydrolysis assays

  • Compare activity rates to those of native IF2

• 30S binding assays:

  • Use filter binding assays to quantify IF2 binding to 30S subunits

  • Apply fluorescence anisotropy with labeled IF2 to measure binding kinetics

  • Perform sucrose gradient centrifugation to isolate 30S-IF2 complexes

• In vitro translation systems:

  • Prepare translation extracts from IF2-depleted cells

  • Add purified recombinant IF2 to these extracts

  • Measure translation of reporter mRNAs

  • Compare activity to systems complemented with native IF2

• Functional complementation:

  • Transform IF2-depleted strains with plasmids expressing recombinant IF2

  • Assess growth restoration as a measure of functional activity

  • Compare growth rates with strains containing native IF2

A comprehensive approach combining multiple methods provides the most reliable assessment of IF2 functional activity.

How can researchers address the challenges of studying temperature-dependent regulation of infB expression?

Studying temperature-dependent regulation of infB expression presents unique challenges that can be addressed through the following approaches:

• RNA stability analysis:

  • Use rifampicin to inhibit transcription initiation at different temperatures

  • Extract RNA at timed intervals to determine decay rates

  • Perform northern blot or qRT-PCR to quantify infB mRNA levels

  • Compare half-lives at different temperatures (e.g., 37°C vs. 10°C)

• Transcriptional analysis:

  • Create transcriptional fusions with reporter genes (e.g., cat gene in pKK232-8)

  • Use primer pairs targeting different regions of the infB promoter

  • Measure reporter activity at different temperatures

  • Identify temperature-responsive promoter elements

• In vitro translation studies:

  • Prepare cell extracts from cultures grown at different temperatures:

    • Non-cold-shock S30 (ncs S30) from cells grown at 37°C

    • Cold-shock S30 (cs S30) from cells after 90 min at 10°C

  • Use these extracts for in vitro translation of infB mRNA

  • Incorporate radioactive amino acids (e.g., [35S] methionine) to track translation products

  • Analyze products by SDS-PAGE, autoradiography, and western blotting

• Temperature shift protocols:

  • Develop consistent protocols for temperature shifts (e.g., 37°C to 10°C)

  • Monitor cellular responses at multiple time points after the shift

  • Consider the impact of cooling rate on cellular responses

  • Include appropriate controls at constant temperatures

These methodologies provide a comprehensive toolkit for investigating the complex relationship between temperature and infB expression.

What are the implications of the conformational switch in IF2 for antibiotic development?

The conformational switch in IF2 presents a promising target for novel antibiotic development:

• Domain III as a druggable target:

  • Recent studies have revealed that domain III of IF2 plays a pivotal, allosteric role in IF2 activation

  • This finding suggests that domain III can be specifically targeted for the development of novel antibiotics

  • Compounds that interfere with this allosteric mechanism could inhibit bacterial translation initiation

• Conformational targeting strategies:

  • Drugs could be designed to:

    • Stabilize inactive conformations of IF2

    • Prevent the GTP-dependent conformational switch

    • Interfere with the interaction between IF2 and fMet-tRNA fMet

    • Disrupt the proper positioning of ribosomal subunits by IF2

• Advantages of targeting IF2:

  • Essential for bacterial translation initiation

  • Structurally distinct from eukaryotic initiation factors

  • Involved in a critical conformational switch mechanism

  • Contains domains highly conserved within bacterial species

• Potential screening approaches:

  • High-throughput screening using FRET-based conformational sensors

  • Structure-based virtual screening targeting domain III

  • Fragment-based drug discovery approaches

  • Phenotypic screens for compounds that inhibit translation initiation

These approaches could lead to a new class of antibiotics targeting a fundamental process in bacterial protein synthesis.

How does current research on IF2 inform our understanding of bacterial translation regulation under stress conditions?

Research on IF2 provides crucial insights into translation regulation under stress conditions:

• Temperature stress response:

  • Studies have shown that temperature shifts (37°C → 10°C) trigger de novo infB expression

  • This response involves both transcriptional and post-transcriptional events

  • Cold shock alters infB mRNA stability and translation efficiency

  • These findings illuminate how bacteria adjust translation initiation during temperature stress

• Nutrient limitation response:

  • IF2 function is linked to the availability of initiator tRNA and GTP

  • Under nutrient limitation, these components may become limiting factors

  • Research into how IF2 activity is modulated during nutrient stress provides insights into bacterial adaptation

• Integration with stress response pathways:

  • IF2 function may be coordinated with other stress response pathways

  • Research on the interplay between IF2 and stress-induced factors helps understand the global regulation of translation

  • These studies reveal how bacteria prioritize protein synthesis during stress

• Translational fidelity under stress:

  • IF2's role in ensuring the fidelity of translation initiation may be particularly important under stress conditions

  • Research on how stress affects IF2's selectivity for initiator tRNA informs our understanding of translational quality control mechanisms

These insights contribute to our broader understanding of bacterial adaptation strategies and may inform approaches to combat bacterial infections.

What are the potential applications of E. coli IF2 in developing expression systems for heterologous proteins?

E. coli IF2 offers several promising applications for improving heterologous protein expression systems:

• Enhanced translation initiation:

  • Co-expression of optimized IF2 variants could improve translation initiation efficiency

  • This approach may be particularly valuable for proteins with non-optimal initiation regions

  • Modifications to IF2 could potentially enhance recognition of non-canonical start codons

• Cold-adapted expression systems:

  • Leveraging the cold-shock response of infB

  • Development of expression systems that utilize cold-inducible promoters derived from infB regulatory regions

  • These systems could facilitate expression of proteins that are toxic or prone to aggregation at higher temperatures

• Stress-responsive expression control:

  • Creating regulatory circuits based on infB stress response elements

  • These systems could allow for fine-tuned expression in response to specific environmental conditions

  • Integration with other stress-responsive elements for sophisticated expression control

• Synthetic biology applications:

  • Developing synthetic IF2 variants with altered specificity or activity

  • Creating orthogonal translation initiation systems with modified IF2 that recognize specific mRNA features

  • These systems could enable selective translation of target mRNAs in complex mixtures

These applications represent the translation of fundamental research on IF2 into practical biotechnological tools that could advance protein production for research and industrial purposes.