Recombinant Alteromonas macleodii Translation initiation factor IF-2 (infB), partial
Description
Definition and Expression
Recombinant A. macleodii IF-2 (infB), partial refers to a truncated, engineered version of the IF-2 protein expressed in heterologous systems (e.g., E. coli). The "partial" designation indicates that only a specific domain or region of the full-length IF-2 is produced, typically corresponding to functional subdomains such as the C-terminal fMet-tRNA binding region (IF2-C2) or GTPase domains (IF2-G2/G3) .
Key Features:
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Expression System: Likely produced via plasmid-based overexpression in E. coli, followed by affinity purification.
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Sequence Coverage: Partial sequences often retain critical functional motifs, such as the ribosome-binding or tRNA-interaction regions .
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Molecular Weight: Based on homologous systems (e.g., B. stearothermophilus), partial IF-2 fragments range between 20–40 kDa, depending on the truncated region .
Table 1: Predicted Domains in Partial A. macleodii IF-2
| Domain | Function | Homology to B. stearothermophilus |
|---|---|---|
| IF2-G2/G3 | GTP hydrolysis, ribosome binding | 85% sequence similarity |
| IF2-C2 | fMet-tRNA binding | 78% sequence similarity |
Note: Structural data for A. macleodii IF-2 is inferred from homologous systems due to limited direct studies.
Functional Roles
Partial IF-2 retains critical activities essential for in vitro studies:
Table 2: Key Functional Properties
4.1. Domain Flexibility and tRNA Positioning
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The IF2-C2 domain exhibits conformational independence from the G-domain, enabling dynamic tRNA adjustment during initiation .
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GTP binding induces structural rearrangements in IF2-G2, but these changes are not propagated to the C2 domain, suggesting modular functionality .
4.2. Kinetic Binding Hierarchy
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IF2·GTP binds the 30S ribosomal subunit before fMet-tRNA, contradicting earlier carrier hypotheses .
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Rate constants for IF2-30S binding:
Applications and Implications
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Structural Biology: Partial IF-2 fragments enable crystallographic studies of domain-specific interactions .
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Antibiotic Development: Targeting IF2’s GTPase or tRNA-binding domains could disrupt bacterial translation .
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Evolutionary Insights: Comparative studies with archaeal homologs (e.g., aIF5B) highlight functional divergence in tRNA handling .
Challenges and Future Directions
Product Specs
Target Background
KEGG: amc:MADE_1008920
Q&A
Structural and Functional Characterization of Partial IF-2 Domains
Question: How do truncated domains of A. macleodii IF-2 (e.g., GTPase or tRNA-binding regions) retain functional activity compared to full-length IF2?
Answer: Partial IF-2 fragments (e.g., G2/G3 domains for GTP hydrolysis or C2 domain for fMet-tRNA binding) exhibit modular functionality. Structural studies in homologous systems (B. stearothermophilus) reveal that GTP binding induces conformational changes in the G-domain, while the C2 domain remains structurally independent, enabling dynamic tRNA adjustment.
| Domain | Function | Key Features | Experimental Evidence |
|---|---|---|---|
| G2/G3 | GTP hydrolysis | GTP-binding motifs (e.g., Walker A/B) | NMR studies of G2 domain dynamics |
| C2 | fMet-tRNA binding | Ribonucleoprotein interactions | FRET assays in B. stearothermophilus |
Data Contradiction: Early hypotheses suggested IF2 acts as a "carrier" for tRNA, but recent studies show IF2 binds the 30S subunit before fMet-tRNA, challenging this model.
Experimental Approaches to Study IF-2’s Role in Translation Initiation
Question: What methodologies are optimal for analyzing the role of recombinant A. macleodii IF-2 in ribosome function?
Answer: Multi-scale approaches are required:
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In vitro assays: Kinetic binding assays to measure IF2-30S subunit association (e.g., rate constants for IF2·GTP binding).
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Structural biology: Cryo-EM of 70S initiation complexes to resolve conformational changes in IF2-GDP vs. IF2-GTP states .
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Functional complementation: Deletion of N-terminal regions (e.g., PXXXAP repeats in Stigmatella aurantiaca) increases complementation efficiency in E. coli mutants, suggesting structural flexibility enhances function .
Functional Implications of N-Terminal Sequence Deletion
Question: Why does deletion of the N-terminal PXXXAP repeat in Stigmatella aurantiaca IF2 enhance complementation in E. coli mutants?
Answer: The N-terminal region (160 residues, rich in alanine/proline) may act as a regulatory domain. Its deletion eliminates steric hindrance, allowing tighter binding to E. coli ribosomes. Cross-reactivity with E. coli antiserum indicates conserved G-domain/C-terminal regions drive functional complementarity despite N-terminal divergence .
Mechanism of IF2-GDP Conformational Changes
Question: How does GTP hydrolysis to GDP induce IF2 conformational rearrangements critical for tRNA accommodation?
Answer: Cryo-EM structures reveal that GDP binding unlocks a cascade of movements:
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G-domain rotation: Switch 2 α-helix reorientation in IF2-GDP.
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C2-domain displacement: 35 Å movement away from tRNA, releasing it into the P site .
This mechanism explains how IF2 gates the transition to elongation-competent ribosomes.
Distinguishing IF2α and IF2β Forms
Question: How do researchers differentiate between IF2α (full-length) and IF2β (N-terminally truncated) in A. macleodii?
Answer:
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N-terminal sequencing: Edman degradation confirms distinct sequences for IF2α and IF2β, aligning with dual initiation sites in the infB gene .
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Fusion proteins: LacZ fusions reveal IF2α-β-galactosidase (170 kDa) and IF2β-β-galactosidase (150 kDa), indicating independent translation initiation .
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Ribosomal binding: Deletion of the Shine-Dalgarno sequence upstream of the IF2α start site abolishes IF2α production, confirming separate ribosome recruitment .
Optimizing Recombinant Expression of Partial IF-2
Question: What challenges arise when expressing partial IF-2 domains in heterologous systems (e.g., E. coli)?
Answer:
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Protein solubility: C2 domains may misfold due to hydrophobic regions; co-expression with chaperones (e.g., DnaK) improves yield.
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Domain truncation: Truncation boundaries must preserve critical motifs (e.g., Walker A/B in G2).
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Purification: Affinity tags (e.g., His6) are placed at termini to avoid disrupting domain interactions.
Addressing Data Contradictions in IF2 Studies
Question: How do conflicting reports on IF2’s role in subunit joining vs. tRNA binding get resolved?
Answer:
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Kinetic assays: Quantify IF2·GTP binding rates to 30S subunits vs. fMet-tRNA.
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Structural validation: Cryo-EM of IF2-GTP vs. GDP states clarifies conformational states.
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Comparative genomics: Phylogenetic analysis of IF2 sequences across Alteromonas ecotypes may reveal adaptive changes in domain organization .
Environmental Adaptation of IF2 in Alteromonas Ecotypes
Question: Does the partial IF-2 structure correlate with ecological niches in A. macleodii?
Answer:
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Deep-sea vs. surface ecotypes: Comparative genomics shows divergent translation factor repertoires, potentially affecting ribosome efficiency under low-energy conditions .
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GTPase activity: High-energy environments may favor IF2 variants with enhanced GTP hydrolysis rates.
Methodological Limitations in IF2 Research
Question: What technical gaps remain in studying partial IF-2 function?
Answer:
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Dynamic studies: Real-time monitoring of IF2 conformational changes during initiation is limited to end-point assays.
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In vivo relevance: In vitro results (e.g., complementation) may not fully replicate in vivo translation contexts .
Future Directions
Question: How can recombinant IF-2 fragments advance mechanistic studies of translation initiation?
Answer:
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Single-molecule FRET: Track IF2·GTP/GDP conformational dynamics.
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Cryo-EM of mutant complexes: Test hypotheses about domain interactions.
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Comparative studies: Contrast A. macleodii IF2 with extremophiles (e.g., B. stearothermophilus) to identify adaptive residues.
