The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers.
Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.
Grkovic
S
S
School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Brown
M H
MH
Schumacher
M A
MA
Brennan
R G
RG
Skurray
R A
RA
D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides; D018432:Drug Resistance, Multiple / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics*
D001426:Bacterial Proteins; D004220:Disulfides; D026901:Membrane Transport Proteins; C442128:QacR protein, Staphylococcus aureus; D012097:Repressor Proteins; C068498:qacA protein, Staphylococcus aureus; D003545:Cysteine