Energetics of the sequence-specific binding of single-stranded DNA by the F factor relaxase domain.
Transfer of conjugative plasmids between bacteria requires the activity of relaxases or mobilization proteins. These proteins nick the plasmid in a site- and strand-specific manner prior to transfer of the cut strand from donor to recipient. TraI36, the relaxase domain of TraI from plasmid F factor, binds a single-stranded DNA (ssDNA) oligonucleotide containing an F factor sequence with high affinity and sequence specificity. To better understand the energetics of this interaction, we examined the temperature, salt, and pH dependence of TraI36 recognition. Binding is entropically driven below 25 degrees C and enthalpically driven at higher temperatures. van't Hoff analysis yields an estimated deltaC(P)(0) of binding (-3300 cal x mol(-1) x K(-1)) that is larger and more negative than that observed for most double-stranded DNA (dsDNA)-binding proteins. Based on analyses of circular dichroism data and the crystal structure of the unliganded protein, we attribute the deltaC(P)(0) to both burial of hydrophobic surface area and coupled folding and binding of the protein. The salt dependence of the binding indicates that several ssDNA phosphates are buried in the complex, and the pH dependence of the binding suggests that some of these ssDNA phosphates form ionic interactions with basic residues of the protein. Although data are available for relatively few sequence-specific ssDNA-binding proteins, sufficient differences exist between TraI36 and other proteins to indicate that, like dsDNA-binding proteins, ssDNA-binding proteins use different motifs and combinations of forces to achieve specific recognition.
Stern
Jennifer C
JC
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Anderson
Brian J
BJ
Owens
Thomas J
TJ
Schildbach
Joel F
JF
D016880:Anisotropy; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005144:F Factor / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry; D013696:Temperature
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012492:Salts; C027093:TraI protein, E coli; D004265:DNA Helicases