Recombinant Protein traI (traI)
Description
DNA Processing
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Relaxase activity: Binds single-stranded oriT DNA with high specificity (affinity reduced 8,000-fold by single-base mutations) .
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Helicase activity: Unwinds plasmid DNA at rates exceeding 500 bp/s, powered by ATP hydrolysis .
Functional Interactions
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Forms a relaxosome complex with TraY and host factor IHF at oriT .
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Overexpression in trans represses conjugation by 100-fold, suggesting a role in sequestering conjugative components .
Mutagenesis and Functional Analysis
A mutagenesis screen inserting 31-codon sequences into TraI identified regions critical for conjugation:
| Insertion Site | Conjugation Efficiency (% of Wild-Type) | Functional Domain Affected |
|---|---|---|
| Q6 (Near N-terminus) | 77% | Relaxase |
| Q369 (Central region) | 99% | Regulatory |
| V1130 (Helicase domain) | 84% | Helicase |
| L1753 (C-terminus) | 18% | Export interaction |
Data adapted from Matson et al. (2006)
Mutants in the central region (residues 369–683) showed no helicase or relaxase defects but abolished TraI’s repressive effect when overexpressed, implicating this region in regulatory interactions .
DNA Binding and Specificity
Electrophoretic mobility shift assays (EMSAs) revealed:
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Full-length TraI binds oriT DNA with higher affinity (K<sub>d</sub> ~50 nM) than isolated domains .
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The relaxase domain (TraI36) recognizes an 11-bp oriT sequence, with single mismatches reducing binding affinity by up to 10<sup>3</sup>-fold .
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Random DNA sequences show no significant binding below 400 nM TraI .
Applications and Research Implications
Recombinant TraI is used to:
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Study conjugative plasmid transfer mechanisms.
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Develop inhibitors targeting relaxase/helicase activities to combat antibiotic resistance spread.
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Engineer synthetic DNA transfer systems in biotechnology.
Key Strains and Plasmids in TraI Studies
| Strain/Plasmid | Description | Utility |
|---|---|---|
| p99I+ | pTrc99A vector expressing wild-type TraI | Complementation assays |
| F′ΔI | F-plasmid with traI replaced by tetracycline cassette | Genetic knockout studies |
| pLOW2traM0oriT | oriT-containing plasmid with disabled traM | In vitro relaxase activity assays |
Data compiled from Matson et al. (2006) and Lara et al. (2023)
Open Questions and Future Directions
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How does the C-terminal domain interface with the type IV secretion system?
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Can TraI’s helicase activity be harnessed for nucleic acid amplification technologies?
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Do TraI homologs in other plasmids share similar regulatory mechanisms?
