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DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA
1Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry
|September 19, 2001
Summary
The TraI protein precisely binds and nicks DNA at the origin of transfer (oriT), a crucial step for F Factor plasmid transfer. Its high sequence specificity suggests a key regulatory role in initiating and terminating this process.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The TraI protein is essential for conjugative plasmid F Factor transfer.
- TraI possesses dual functions: DNA nicking at the origin of transfer (oriT) and helicase activity.
- TraI binds and cleaves single-stranded DNA oligonucleotides containing the oriT sequence.
Purpose of the Study:
- To investigate the sequence specificity of TraI's DNA recognition at the oriT.
- To determine how TraI binding affinity to oriT variants affects plasmid mobilization efficiency.
Main Methods:
- Used TraI36, a domain of TraI with nicking activity, to measure binding to oriT oligonucleotide variants.
- Assessed binding affinities through quantitative measurements.
- Correlated in vitro binding data with in vivo plasmid mobilization frequencies.
Main Results:
- TraI36 exhibits high sequence specificity for an 11-base region within oriT.
- Single base changes in oriT can decrease TraI binding affinity by up to 8000-fold.
- Lower TraI binding affinities correlate with reduced plasmid transfer efficiencies between cells.
- Efficient nicking and mobilization require a pyrimidine nucleotide upstream of the nick site.
Conclusions:
- TraI's high sequence specificity for oriT, even in single-stranded DNA, indicates a significant regulatory role in plasmid transfer initiation and termination.
- The findings elucidate the molecular mechanisms underlying conjugative plasmid transfer and TraI's function.