Single-stranded DNA binding by F TraI relaxase and helicase domains is coordinately regulated.
Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal approximately 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.
Dostál
Lubomír
L
Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Schildbach
Joel F
JF
D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase; C027093:TraI protein, E coli; D004265:DNA Helicases