Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation.
TrwB is the conjugative coupling protein of plasmid R388. TrwBDeltaN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBDeltaN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K(s) = 8.7 microM) but did not show measurable ATPase or GTPase activity. A single ATP binding site was found per TrwB monomer. An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBDeltaN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBDeltaN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA around nic. The mutant protein TrwB(K136T)DeltaN70 was indistinguishable from TrwBDeltaN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.
Moncalián
G
G
Departamento de Biología Molecular (Unidad Asociada al Centro de Investigaciones Biológicas), Universidad de Cantabria, C/Herrera Oria s/n, 39011 Santander, Spain.
Cabezón
E
E
Alkorta
I
I
Valle
M
M
Moro
F
F
Valpuesta
J M
JM
Goñi
F M
FM
de La Cruz
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D010957:Plasmids; D011485:Protein Binding
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C403125:trwB protein, E coli; D000255:Adenosine Triphosphate