Recombinant Translation initiation factor IF-3 (infC)

What is the basic domain organization of bacterial Translation Initiation Factor 3?

Translation Initiation Factor 3 consists of two domains - the C-terminal domain (IF3C) and the N-terminal domain (IF3N) - connected by a hydrophilic, lysine-rich linker. Each domain can independently bind to the 30S ribosomal subunit and occupies different binding sites during various stages of translation initiation. The C-terminal domain can interact with either the P site (C2 position) or with helix 45 and helix 24 (C1 position). Similarly, the N-terminal domain can bind either near uS11 on the 30S platform (NR position) or on the elbow of initiator tRNA (NT position) .

What are the primary functions of IF3 in bacterial translation initiation?

IF3 performs three critical functions during translation initiation:

• It prevents premature association of the 50S ribosomal subunit by blocking the formation of inter-subunit bridges B2a and B2b

• It enhances the rate of P site codon-anticodon interaction between fMet-tRNAfMet and the initiation triplet of mRNA, improving start codon selection

• It orchestrates a kinetic checkpoint that determines when the ribosome can enter the elongation phase of protein synthesis

These functions collectively enhance the fidelity and speed of bacterial translation initiation .

How does IF3 contribute to translation initiation fidelity?

IF3 enhances translation fidelity by:

• Preventing non-initiator tRNAs from being used during initiation

• Debarring the use of non-canonical start codons

• Increasing the dissociation rate of non-canonical and pseudo-30S initiation complexes

• Preventing premature 50S subunit joining, which gives time for proper initiation complex formation

• Enhancing the interaction between the initiator tRNA anticodon and the start codon in the P site

The C-terminal domain (IF3C) positioned at the C2 site near the P site plays a particularly important role in start codon selection .

What techniques are used to study IF3 dynamics during translation initiation?

Several techniques are employed to study IF3 dynamics:

• Förster Resonance Energy Transfer (FRET): Using double-labeled IF3 (IF3DL) with donor and acceptor fluorophores to monitor interdomain movements in real-time. In particular, a fluorescent derivative with donor and silent acceptor dyes specifically linked to the N and C terminal domains allows researchers to track conformational changes.

• Pre-steady state kinetics: Combined with FRET to determine rates of conformational changes during various stages of initiation.

• Molecular modeling: Based on available structures from cryo-EM and X-ray crystallography to interpret FRET data in structural context.

• Time-resolved chemical probing: To monitor binding events and conformational changes.

• Single-molecule FRET: To observe individual molecular events rather than ensemble averages .

How can researchers prepare functional double-labeled IF3 for FRET studies?

To prepare double-labeled IF3 (IF3DL) for FRET studies:

• Introduce minimal mutagenesis to IF3, such as modifying a single amino acid position (E166C) that is solvent-exposed in both free and 30S-bound IF3.

• Choose positions that don't disturb secondary structures of the domains - for example, in published studies, a cysteine at position 166 in IF3C was selected because it doesn't disturb any secondary structure.

• Utilize the wild-type cysteine at position 65 with fluorophores having five-carbon linkers to reduce potential interference with the IF3N structure.

• Label the domains with donor and acceptor fluorophores (or silent acceptor for certain studies) that allow for FRET measurements.

• Verify that the labeled IF3 maintains its biological activity through functional assays.

This approach allows monitoring of intramolecular FRET changes resulting from movements of IF3 domains relative to each other .

What kinetic measurements can reveal IF3 conformational changes during translation initiation?

Kinetic measurements using stopped-flow apparatus can reveal:

• The timing and rates of IF3 domain rearrangements in response to binding of other initiation factors (IF1, IF2)

• Changes in IF3 conformation upon fMet-tRNAfMet recruitment

• The effect of mRNA binding and start codon recognition on IF3 positioning

• Conformational changes associated with 50S subunit joining and IF3 dissociation

In these experiments, an increase in donor fluorescence over time indicates that IF3 domains are moving apart, while a decrease suggests the domains are getting closer together. By varying the concentration of binding partners and measuring the resulting rates, researchers can determine whether observed conformational changes correspond to binding events or subsequent rearrangements .

What is the sequence of conformational changes IF3 undergoes during translation initiation?

IF3 undergoes a series of distinct conformational changes during translation initiation:

• Initial binding to the 30S subunit: IF3 binds with domains in an extended conformation.

• Response to IF1 and IF2 binding: IF3 domains move closer together, with IF3C moving toward the P site (C2 position) and IF3N becoming more dynamic.

• fMet-tRNAfMet binding: Initially causes IF3 domains to move apart as IF3N is transiently displaced toward the E site.

• Start codon recognition: Leads to IF3C displacement from C2 toward the C1 site, which is the slowest step (rate-limiting) in 30S initiation complex formation.

• 50S subunit joining: Causes IF3 domains to come together prior to complete dissociation from the ribosome.

• Recycling: IF3 is released and becomes available for a new round of translation initiation .

How do other initiation factors influence IF3 dynamics?

Other initiation factors significantly impact IF3 conformational dynamics:

• IF1 binding promotes IF3 compaction and movement of IF3C toward the P site (C2 position), with observed rate constants of approximately 2.5 s-1.

• IF2 alone has a similar but less pronounced effect on IF3 conformation.

• Together, IF1 and IF2 have a cooperative effect, maximizing the compaction of IF3 and the positioning of IF3C near the P site. This cooperative effect enhances the anti-association activity of IF3.

• The presence of these factors helps create a conformational state of IF3 that is optimized for initiator tRNA binding, with IF3N creating a "pocket" ready to accept the initiator tRNA.

These interactions occur relatively quickly, with the 30S-IFs complex assembling in approximately 30 ms and rearranging in about 1 second .

What role does the initiator tRNA play in IF3 conformational dynamics?

The initiator tRNA (fMet-tRNAfMet) plays several key roles in IF3 dynamics:

• When fMet-tRNAfMet binds to a 30S complex containing IFs, it causes IF3 domains to move apart from each other, indicating a single-step re-accommodation of IF3 domains.

• The kinetics of this interaction show a linear dependence on tRNA concentration, consistent with IF3 monitoring the initial bimolecular interaction between fMet-tRNAfMet and the 30S pre-initiation complex.

• The formyl group of the initiator tRNA is particularly important - when absent (using Met-tRNAfMet instead), IF3 does not undergo the same conformational changes.

• Upon binding, fMet-tRNAfMet transiently displaces IF3N toward the E site, occupying the NT binding sites.

• In later steps, the proper positioning of fMet-tRNAfMet in the P site and correct codon-anticodon interaction leads to IF3C displacement from C2 to C1, which is the rate-limiting step in translation initiation .

How can researchers distinguish between functional and non-functional 30S initiation complexes using IF3?

Researchers can distinguish between functional and non-functional 30S initiation complexes by:

• Monitoring IF3 conformational changes using FRET-based assays with double-labeled IF3. In functional complexes with correct start codons and initiator tRNA, IF3 undergoes a characteristic sequence of conformational changes ending with IF3C displacement and eventual factor dissociation.

• Comparing kinetic rates: In non-functional complexes (e.g., those lacking an initiation codon), IF3 responds to fMet-tRNAfMet binding but fails to undergo complete displacement and dissociation.

• Testing with altered components: When using mRNAs lacking initiation codons or non-formylated initiator tRNAs, IF3 does not exhibit the same pattern of conformational changes observed with canonical components.

• Measuring 50S subunit joining rates: In proper initiation complexes, IF3 displacement allows efficient 50S joining, which can be monitored with suitable fluorescent labels.

These approaches allow researchers to use IF3 as a reporter for initiation complex quality .

What are the key considerations when designing experiments to study IF3 interactions with non-canonical initiation complexes?

When designing experiments to study IF3 interactions with non-canonical initiation complexes:

• Component selection:

  • Use specific non-canonical components (e.g., mRNAs with non-AUG start codons, non-initiator tRNAs, or initiator tRNAs lacking formylation)

  • Ensure all other components are highly purified and functional

• Detection strategy:

  • Employ double-labeled IF3 for FRET measurements

  • Consider multiple measurement approaches (bulk FRET, single-molecule FRET, chemical probing)

  • Include positive and negative controls with canonical components

• Kinetic analysis:

  • Design pre-steady state experiments to capture transient states

  • Compare rates with canonical initiation to identify specific steps affected

  • Use concentration dependency to distinguish binding from conformational events

• Data interpretation:

  • Correlate FRET changes with structural models

  • Consider that IF3 may adopt alternative conformations not seen in canonical initiation

  • Be aware that altered kinetics rather than complete inhibition may be observed

• Validation:

  • Verify findings with multiple experimental approaches

  • Correlate in vitro observations with in vivo effects when possible .

How do mutations in the linker region between IF3N and IF3C domains affect its function?

Mutations in the linker region between IF3 domains can significantly impact function:

• Length alterations: Shortening the linker connecting IF3C to IF3N has been shown to result in incorrect decoding of the mRNA start site and fitness loss in vivo. This suggests that the proper spacing between domains is critical for IF3 to simultaneously interact with different parts of the ribosome and initiator tRNA.

• Flexibility changes: Mutations that alter the flexibility of the lysine-rich linker can affect the dynamic movement of IF3 domains and their ability to adopt different conformations during initiation.

• Charge modifications: Since the linker is hydrophilic and lysine-rich, changing the charge characteristics can alter interactions with the negatively charged ribosomal RNA.

• Functional consequences: Linker mutations primarily affect:

  • The ability of IF3 to enhance start codon selection

  • The anti-association activity that prevents premature 50S joining

  • The kinetic checkpoint function in late stages of initiation

These observations underscore the importance of not just the domains themselves but also their spatial relationship maintained by the linker .

How does understanding IF3 dynamics contribute to developing new antibiotics?

Understanding IF3 dynamics offers several avenues for antibiotic development:

• Novel target identification: Since IF3 is essential for bacterial translation initiation but structurally and functionally distinct from eukaryotic initiation factors, it represents a potential target for bacteria-specific antibiotics.

• Mechanism-based design: Knowledge of specific binding sites and conformational changes allows for the design of compounds that:

  • Lock IF3 in non-functional conformations

  • Prevent proper interaction with the 30S subunit

  • Disrupt the dynamic cycle required for function

• Screening approaches: Research on IF3 dynamics enables the development of high-throughput screening assays based on:

  • FRET signals from labeled IF3

  • Functional assays measuring translation initiation efficiency

  • Displacement of IF3 from ribosomal complexes

• Species specificity: IF3 has some sequence and structural variations between bacterial species, offering potential for narrow-spectrum antibiotics targeting specific pathogens.

• Resistance considerations: Understanding the complete functional cycle helps predict potential resistance mechanisms and design strategies to overcome them .

What experimental approaches can be used to study species-specific differences in IF3 function?

To study species-specific differences in IF3 function, researchers can employ:

• Comparative sequence and structure analysis:

  • Align IF3 sequences from different bacterial species

  • Model species-specific IF3 structures based on available data

  • Identify conserved vs. variable regions that might confer functional differences

• Heterologous expression systems:

  • Express recombinant IF3 from different bacterial species

  • Purify and label for functional and conformational studies

  • Test cross-species compatibility by mixing components from different bacteria

• Domain-swapping experiments:

  • Create chimeric IF3 proteins with domains from different species

  • Assess which domains confer species-specific properties

  • Identify critical residues through targeted mutagenesis

• Comparative functional assays:

  • Measure translation initiation rates with species-matched vs. mismatched components

  • Assess fidelity functions across species using non-canonical initiation elements

  • Compare binding affinities to ribosomes from different bacterial sources

• In vivo complementation studies:

  • Test whether IF3 from one species can complement deletion in another

  • Identify functional deficits when using heterologous IF3

  • Create reporter systems to quantify initiation efficiency and fidelity .

How can IF3 be used as a tool to study ribosomal assembly and function?

IF3 can serve as a powerful tool for studying ribosomal assembly and function:

• As a conformational probe:

  • Double-labeled IF3 can report on ribosome states during initiation

  • Changes in IF3 FRET signal indicate specific steps in ribosomal complex formation

  • The kinetics of IF3 conformational changes can reveal rate-limiting steps

• For purification of initiation complexes:

  • IF3-bound 30S subunits can be isolated to study pre-initiation states

  • The factor prevents 50S joining, allowing accumulation of 30S initiation complexes

  • Tagged versions can be used for pull-down experiments

• To study ribosome heterogeneity:

  • IF3 binding and dynamics may differ on specialized ribosomes

  • The factor can reveal structural variations in the platform and P-site regions

  • Differences in IF3 behavior can indicate functional specialization of ribosomes

• As a quality control tool:

  • IF3 preferentially binds to properly matured 30S subunits

  • Its binding patterns can indicate defects in ribosome assembly

  • The factor helps discriminate between functional and non-functional ribosomes

• To study antibiotic effects:

  • Changes in IF3 dynamics in the presence of antibiotics can reveal mechanisms of action

  • The factor can be used to identify compounds targeting specific steps in initiation

  • IF3-based assays can screen for inhibitors of translation initiation .