DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA.
The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.
Stern
J C
JC
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Schildbach
J F
JF
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005144:F Factor / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018741:Replication Origin
D017931:DNA Primers; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004720:Endonucleases; C027093:TraI protein, E coli; D004265:DNA Helicases