Recombinant Enterococcus faecalis Translation initiation factor IF-3 (infC)

What is the basic structure of E. faecalis Translation Initiation Factor IF-3?

Translation Initiation Factor IF-3 in E. faecalis, encoded by the infC gene, consists of two distinct domains connected by a flexible linker. Similar to other bacterial IF-3 proteins, it comprises an N-terminal domain (NTD) and a C-terminal domain (CTD), each with specific functional roles. The CTD carries out most of the known functions of IF-3 and can independently sustain bacterial growth, while the NTD plays important roles in interacting with initiator tRNA and coordinating the movement of both domains during the translation initiation pathway .

How does E. faecalis IF-3 differ structurally from other bacterial IF-3 proteins?

While the general two-domain structure of IF-3 is conserved across bacterial species, notable differences exist in E. faecalis compared to model organisms like E. coli. Unlike E. coli IF-3, which is known to utilize an AUU (ATT) start codon for translational regulation, E. faecalis infC is annotated to start with the canonical ATG codon. This difference may impact regulatory mechanisms controlling IF-3 expression levels and subsequent effects on translation initiation efficiency .

What are the primary functions of Translation Initiation Factor IF-3 in E. faecalis?

Translation Initiation Factor IF-3 in E. faecalis serves multiple critical functions:

• Acts as a fidelity factor during assembly of the ternary initiation complex

• Ensures correct recognition of start codons in mRNA

• Facilitates proper pairing of the initiator tRNA anticodon with the start codon in the ribosomal P-site

• Prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex

• Stabilizes free 30S subunits

• Enables 30S subunits to bind to mRNA

• Verifies accuracy against the first aminoacyl-tRNA

• Promotes rapid codon-anticodon pairing for initiator tRNA binding

What are the recommended methods for recombinant expression of E. faecalis IF-3?

For effective recombinant expression of E. faecalis IF-3, researchers should consider the following methodological approach:

• Vector Selection: Choose expression vectors compatible with E. faecalis, such as those based on the pWV01 replicon.

• Optimized Codons: Ensure the coding sequence utilizes codons optimized for E. faecalis expression.

• Promoter Selection: Utilize constitutive promoters like P23 for stable expression or inducible promoters for controlled expression.

• Transformation Strategy: For clinical E. faecalis strains with limited transformation efficiency, consider conjugation using donor strains such as E. faecalis CK111, which contains chromosomally integrated pWV01 repA gene under the control of the lactococcal P23 promoter.

• Selection Markers: Incorporate appropriate antibiotic resistance markers for selection of transformants.

• Expression Verification: Confirm successful expression through Western blotting and functional assays .

How can researchers effectively purify recombinant E. faecalis IF-3 for in vitro studies?

Purification of recombinant E. faecalis IF-3 requires careful consideration of its biochemical properties and typically follows these steps:

• Expression Optimization: Express the recombinant protein with an affinity tag (His-tag or GST-tag) to facilitate purification.

• Cell Lysis: Use optimized lysis buffers (typically containing 20-50 mM Tris-HCl pH 7.5, 100-300 mM NaCl, 5-10% glycerol) with protease inhibitors.

• Affinity Chromatography: Purify using Ni-NTA columns for His-tagged proteins or glutathione resins for GST-tagged proteins.

• Tag Removal: If necessary, remove affinity tags using site-specific proteases.

• Ion Exchange Chromatography: Further purify using anion or cation exchange chromatography based on the protein's isoelectric point.

• Size Exclusion Chromatography: Perform final purification step to remove aggregates and ensure homogeneity.

• Verification: Confirm purity by SDS-PAGE and functionality through activity assays with 30S ribosomal subunits .

What methods can be used to study the interaction between E. faecalis IF-3 and the ribosome?

Several complementary approaches can be employed to study E. faecalis IF-3-ribosome interactions:

• Molecular Dynamics (MD) Simulations: Investigate atomic-level interactions between IF-3 domains and ribosomal components or initiator tRNA.

• Site-Directed Mutagenesis: Introduce mutations at key residues (e.g., R25A/Q33A/R66A in the NTD) to disrupt specific interactions.

• Cryo-Electron Microscopy: Visualize IF-3 binding to the 30S ribosomal subunit at different stages of initiation.

• Fluorescence-Based Assays: Use fluorescently labeled IF-3 to monitor binding kinetics and conformational changes.

• Toe-Printing Assays: Assess the impact of IF-3 on mRNA positioning on the ribosome.

• Filter-Binding Assays: Quantitatively measure IF-3 affinity for ribosomes under various conditions.

• Ribosome Dissociation Assays: Evaluate the role of IF-3 in preventing premature 50S subunit association .

How do the N-terminal and C-terminal domains of E. faecalis IF-3 coordinate during translation initiation?

The coordination between the N-terminal (NTD) and C-terminal (CTD) domains of E. faecalis IF-3 involves a sophisticated interplay crucial for translation initiation fidelity:

What role does E. faecalis IF-3 play in start codon selection and translation initiation fidelity?

E. faecalis IF-3 serves as a critical checkpoint for translation initiation fidelity through multiple mechanisms:

• Start Codon Discrimination: IF-3 monitors the codon-anticodon interaction between mRNA and initiator tRNA, preferentially stabilizing complexes with canonical start codons (primarily AUG in E. faecalis, which accounts for 81.5% of start codons).

• Non-canonical Start Codon Regulation: For non-canonical start codons (GUG: 10%, UUG: 8.5% in E. faecalis), IF-3 modulates initiation efficiency, likely through conformational changes that affect stability of the initiation complex.

• Initiator tRNA Verification: IF-3 specifically verifies features of the initiator tRNA, including the characteristic three consecutive GC base pairs in its anticodon stem that facilitate preferential binding to the ribosomal P-site.

• Dissociation Promotion: When non-cognate tRNAs or incorrect start codons are present, IF-3 increases dissociation of the initiation complex, preventing inappropriate translation initiation.

• 50S Subunit Association Control: IF-3 prevents premature docking of the 50S ribosomal subunit until a correct initiation complex has formed .

How does the genetic context of the infC gene in E. faecalis influence its expression and function?

The genetic context of the infC gene in E. faecalis presents several interesting features that influence its expression and function:

• Start Codon Selection: Unlike E. coli infC, which uses a rare AUU start codon for autoregulation, E. faecalis infC is annotated to use the canonical AUG start codon, potentially impacting its translational regulation mechanisms.

• Genomic Environment: The genomic context of infC in E. faecalis may include nearby genes involved in translation or ribosome biogenesis, creating potential for coordinated regulation.

• Recombination Potential: E. faecalis exhibits high levels of recombination, which may facilitate the acquisition of genetic elements affecting infC expression. This recombination capability contributes to the genetic plasticity of E. faecalis and may influence the evolution of translation initiation mechanisms.

• Regulatory Elements: The promoter region and other regulatory elements affecting infC expression in E. faecalis may differ from those in model organisms, potentially leading to species-specific regulation patterns.

• Horizontal Gene Transfer Impacts: The capability of E. faecalis to participate in horizontal gene transfer, particularly through conjugative plasmids, may influence infC expression if regulatory elements are acquired through such mechanisms .

How can mutations in E. faecalis IF-3 be designed to study translation initiation mechanisms?

Designing strategic mutations in E. faecalis IF-3 provides powerful tools for investigating translation initiation mechanisms:

• Domain-Specific Mutations:

  • C-terminal Domain (CTD): Mutations affecting ribosome binding can isolate the role of CTD in start codon recognition

  • N-terminal Domain (NTD): Targeted mutations (e.g., R25A/Q33A/R66A) can disrupt interactions with initiator tRNA without affecting NTD structure

  • Linker Region: Alterations in the flexible linker can assess its role in coordinating domain movements

• Functional Residue Targeting:

  • Residues involved in RNA binding

  • Residues affecting domain interactions

  • Residues mediating conformational changes

• Experimental Approach:

  • Site-directed mutagenesis to create precise amino acid substitutions

  • Domain swapping between E. faecalis and other bacterial IF-3 proteins

  • Truncation mutations to assess domain-specific functions

• Phenotypic Analysis Methods:

  • Growth rate measurements under various conditions

  • In vitro translation assays with purified components

  • Ribosome binding assays to assess interaction kinetics

  • Structural studies to determine effects on protein conformation

What are the implications of E. faecalis IF-3 research for understanding antibiotic resistance mechanisms?

Research on E. faecalis IF-3 has significant implications for understanding antibiotic resistance mechanisms:

• Translation-Targeting Antibiotics: Many antibiotics (tetracyclines, aminoglycosides, macrolides) target the bacterial translation apparatus. Understanding how IF-3 functions in E. faecalis can reveal potential species-specific vulnerabilities or resistance mechanisms.

• Stress Response Connection: Translation initiation regulation via IF-3 may influence how E. faecalis responds to antibiotic stress, potentially affecting expression of resistance genes.

• Biofilm Formation Impact: IF-3-mediated translation regulation may influence expression of factors involved in biofilm formation, which can contribute to antibiotic tolerance.

• Antibiotic Development Opportunities: Structural and functional differences between E. faecalis IF-3 and human translation factors could present targets for developing selective antibiotics.

• Resistance Gene Expression: Understanding how IF-3 affects start codon selection efficiency could inform models of resistance gene expression under antibiotic pressure, especially for genes using non-canonical start codons .

How does IF-3 in E. faecalis compare to IF-3 in other clinically relevant bacterial species?

Comparative analysis of IF-3 across clinically relevant bacterial species reveals important differences and similarities:

Key differences include:

• Regulatory mechanisms: E. faecalis likely employs different translational regulation strategies compared to E. coli

• Start codon preferences: E. faecalis primarily uses AUG (81.5%), with less frequent use of alternative start codons

• Species-specific interactions: Potential differences in ribosome-binding interfaces and initiator tRNA recognition

• Evolutionary adaptations: Variations that may reflect adaptations to different ecological niches and stress responses

What are common challenges in expressing recombinant E. faecalis IF-3 and how can they be addressed?

Researchers working with recombinant E. faecalis IF-3 commonly encounter several challenges that can be addressed through specific methodological approaches:

• Protein Solubility Issues:

  • Challenge: IF-3 may form inclusion bodies when overexpressed

  • Solution: Optimize expression conditions (lower temperature, reduced induction), use solubility-enhancing fusion tags (SUMO, MBP), or develop refolding protocols from inclusion bodies

• Functional Activity Maintenance:

  • Challenge: Recombinant IF-3 may lose activity during purification

  • Solution: Include stabilizing agents (glycerol, reducing agents), minimize freeze-thaw cycles, and verify activity with functional assays

• Expression Host Selection:

  • Challenge: Choosing appropriate expression systems for E. faecalis proteins

  • Solution: E. coli BL21(DE3) derivatives work well for initial trials; for complex cases, consider gram-positive expression hosts like B. subtilis or L. lactis

• Codon Usage Differences:

  • Challenge: Codon bias may affect expression efficiency

  • Solution: Optimize codons for the expression host or use strains with rare tRNA supplements

• Genetic Manipulation:

  • Challenge: Transformation efficiency limitations in E. faecalis

  • Solution: Consider conjugation protocols using donor strains like E. faecalis CK111, which contains chromosomally integrated pWV01 repA gene under control of the lactococcal P23 promoter

How can researchers troubleshoot artifacts in E. faecalis IF-3 functional assays?

When conducting functional assays with E. faecalis IF-3, researchers should consider these troubleshooting approaches for common artifacts:

• Non-specific Binding in Ribosome Binding Assays:

  • Artifact: High background signal suggesting non-specific binding

  • Troubleshooting: Adjust salt concentration (typically 100-300 mM), include competitors like BSA or tRNA, and perform careful controls with mutated IF-3 variants

• Domain Misfolding Detection:

  • Artifact: Unexpected functional outcomes due to improper domain folding

  • Troubleshooting: Verify structural integrity through circular dichroism, thermal shift assays, or limited proteolysis before functional testing

• Aggregation During Assays:

  • Artifact: Progressive loss of activity during experiments

  • Troubleshooting: Include stabilizing agents, perform size exclusion chromatography immediately before assays, and monitor protein stability with dynamic light scattering

• Ribosomal Preparation Variability:

  • Artifact: Inconsistent results between different ribosome preparations

  • Troubleshooting: Standardize ribosome isolation protocols, verify ribosome quality through activity assays, and include internal controls

• Buffer Composition Effects:

  • Artifact: Activity variations due to buffer components

  • Troubleshooting: Systematically test the impact of buffer components (Mg²⁺ concentration is particularly critical), pH, and additives on IF-3 activity

What are promising areas for future research on E. faecalis IF-3?

Several promising research directions could significantly advance our understanding of E. faecalis IF-3:

• Structural Dynamics Investigation:

  • High-resolution structural studies of E. faecalis IF-3 in complex with the 30S ribosomal subunit

  • Time-resolved cryo-EM to capture different states during translation initiation

  • Computational modeling of domain movements during the initiation process

• Regulatory Network Mapping:

  • Comprehensive analysis of factors affecting infC expression in E. faecalis

  • Investigation of potential autoregulatory mechanisms specific to E. faecalis

  • Systems biology approaches to place IF-3 within the broader translational control network

• Start Codon Selection Mechanisms:

  • Detailed comparison of E. faecalis IF-3 discrimination between canonical (AUG) and non-canonical (GUG, UUG) start codons

  • Investigation of potential leaderless mRNA translation mechanisms in E. faecalis

  • Analysis of potential specialized translation initiation during stress conditions

• Novel Therapeutics Development:

  • Screening for small molecules that specifically target E. faecalis IF-3

  • Structure-based design of peptides that disrupt IF-3 function

  • Investigation of combination approaches targeting translation initiation

• Evolutionary Adaptation Analysis:

  • Comparative genomics of IF-3 across Enterococcus species in different ecological niches

  • Experimental evolution studies under antibiotic pressure to identify adaptive changes in IF-3

  • Investigation of horizontal gene transfer events affecting translation initiation

How might structural studies of E. faecalis IF-3 contribute to new antimicrobial development?

Structural studies of E. faecalis IF-3 hold significant potential for antimicrobial development through several promising approaches:

• Structure-Based Drug Design:

  • High-resolution structures of E. faecalis IF-3, particularly in complex with the 30S ribosomal subunit, could reveal unique binding pockets

  • Computational screening against these pockets could identify lead compounds that specifically disrupt IF-3 function

  • Rational design of peptidomimetics targeting the interface between IF-3 domains or between IF-3 and ribosomal components

• Species-Specific Targeting:

  • Detailed structural comparisons between E. faecalis IF-3 and human translation factors could identify bacterial-specific features

  • Development of compounds that selectively bind to bacterial-specific regions, minimizing off-target effects

  • Exploitation of differences in domain organization and linker regions unique to bacterial IF-3

• Resistance Mechanism Circumvention:

  • Structural understanding of how mutations in IF-3 might confer resistance to translation-targeting antibiotics

  • Design of compounds targeting multiple sites simultaneously to reduce resistance development

  • Development of adjuvants that enhance the effectiveness of existing antibiotics by modulating IF-3 function

• Novel Binding Site Identification:

  • Investigation of allosteric sites that might not be directly involved in translation but could regulate IF-3 function

  • Exploration of interfaces between IF-3 and other initiation factors or regulatory molecules

  • Targeting the dynamic conformational changes that occur during the initiation process