Recombinant Desulfovibrio vulgaris Translation initiation factor IF-3 (infC)

What is the structure and function of Desulfovibrio vulgaris Translation initiation factor IF-3?

Translation initiation factor IF-3 in D. vulgaris, like in other bacteria, is a two-domain protein consisting of N-terminal (IF3N) and C-terminal (IF3C) domains connected by a flexible linker. The protein enhances translation fidelity through three primary functions:

• Prevention of premature joining of the 50S subunit by impairing inter-subunit bridges B2a and B2b

• Acceleration of the P site codon-anticodon interaction between initiator tRNA and mRNA

• Orchestration of kinetic checkpoints for the ribosome entering the elongation phase

The dynamic movement of IF3 domains occurs at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. This conformational flexibility is essential for its biological function.

What expression systems are optimal for producing functional recombinant D. vulgaris IF-3?

Multiple expression systems have been developed for recombinant D. vulgaris IF-3 production, each with specific advantages:

The E. coli system is commonly used for basic research applications, while more complex systems may be preferred for specialized applications .

How can isotope labeling of D. vulgaris IF-3 facilitate structural and functional studies?

Isotope labeling enhances structural analysis through techniques like nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry:

• 13C/15N Labeling: Enables detailed NMR studies of protein structure and dynamics

• Selective Labeling: Incorporation of labeled amino acids at specific positions to track domain movements

• Deuteration: Improves signal quality in NMR studies of larger protein complexes

For investigating dynamics similar to those observed in other bacterial IF-3 studies, FRET experiments using strategically placed fluorophores can track interdomain movements during the translation initiation process .

How does the dynamic cycle of IF-3 domains contribute to translation initiation in D. vulgaris?

Based on research with bacterial IF-3, the following dynamic cycle likely occurs in D. vulgaris:

• Initial Binding: IF-3 binds to the 30S subunit with domains in extended conformation

• Compaction Phase: IF1 and IF2 promote IF-3 compaction with the C-terminal domain moving toward the P site

• tRNA Selection: The N-terminal domain creates a pocket that accepts initiator tRNA

• Codon Recognition: Decoding of the start codon displaces the C-terminal domain from the P site

• 70S Formation: Domains move into close proximity before dissociation and recycling

This dynamic cycle ensures accurate initiation by preventing subunit joining until the correct initiator tRNA and start codon are in place.

What techniques are most effective for studying IF-3 interactions with the ribosome in anaerobic bacteria like D. vulgaris?

Studying IF-3 interactions in anaerobic bacteria requires specialized approaches:

• Pre-steady State Kinetics: Measures the velocities of domain movements using stopped-flow fluorescence

• Molecular Modeling: Combines available structures with experimental data to predict domain positions

• Cryo-EM: Captures structural snapshots of initiation complexes under near-native conditions

• Single-Molecule FRET: Tracks real-time movements of labeled IF-3 domains during initiation

These techniques have successfully revealed the kinetic spectrum of IF-3 movements in other bacteria and would be applicable to studying D. vulgaris IF-3 .

How can genetic manipulation techniques be applied to study the role of IF-3 in D. vulgaris?

Recent advances in D. vulgaris genetic manipulation provide tools for studying IF-3 function:

• Electroporation-mediated Transformation: Enables replacement of genes via double-crossover homologous recombination

• Antibiotic Resistance Markers: Allow selection of successfully transformed cells

• Gene Replacement Strategies: Can be used to introduce tagged versions or mutations of the infC gene

These methods have been successfully applied to study other D. vulgaris genes and can be adapted for IF-3 research.

What strategies can overcome protein aggregation issues when working with recombinant D. vulgaris IF-3?

Protein aggregation is a common challenge when working with recombinant proteins from anaerobic bacteria:

• Expression Optimization:

  • Lower induction temperature (16-20°C)

  • Reduced inducer concentration

  • Co-expression with chaperones

• Buffer Optimization:

  • Addition of stabilizing agents (glycerol, arginine)

  • Optimization of salt concentration

  • Inclusion of reducing agents for sulfur-rich proteins

• Purification Approaches:

  • Inclusion of detergents below critical micelle concentration

  • On-column refolding techniques

  • Size-exclusion chromatography to remove aggregates

How can researchers verify the functional activity of purified D. vulgaris IF-3?

Functional verification of IF-3 activity can be performed using several complementary assays:

• 30S Binding Assays: Measuring binding affinity to 30S ribosomal subunits using fluorescence anisotropy

• Anti-association Activity: Monitoring prevention of 70S formation in the presence of IF-3

• tRNA Selection Assays: Evaluating discrimination between initiator and elongator tRNAs

• In vitro Translation: Assessing the ability to support translation initiation in reconstituted systems

These assays provide comprehensive validation of IF-3 functionality across its multiple roles in translation initiation.