DNA-binding and oligomerization studies of the manganese(II) metalloregulatory protein MntR from Bacillus subtilis.
The metalloregulatory protein MntR from Bacillus subtilis acts as a transcriptional regulator of manganese homeostasis. MntR is a member of a subfamily of DtxR-related proteins that perform analogous regulatory functions in a variety of pathogenic organisms. Metal ions activate MntR to bind DNA and repress the transcription of the mntH gene, which encodes for a proton-coupled metal ion transporter. Size-exclusion chromatography and sedimentation equilibrium ultracentrifugation studies show that apo MntR is predominantly a homodimer in solution. Using fluorescence anisotropy measurements, the DNA binding properties of MntR have been examined. In the strict absence of divalent transition metal ions MntR has a low affinity for the mntH control sequence (K(d) > 8.0 microM). However, binding of MntR is stimulated by the presence of Mn(2+) and Cd(2+) to generate high affinity binding with K(d) values of 16.0 and 7.3 nM, respectively. MntR is also shown to bind the mntH control sequence in the presence of other divalent transition metals, including Ni(2+), Cu(2+), and Zn(2+), but with much lower affinity (K(d) approximately 1.3-2.3 microM). The data here demonstrate that differences in metal-activated DNA binding plays a role in the mechanism of manganese(II)-selective transcription factors and that the oligomerization of MntR is metal-independent, which distinguishes this protein from iron(II)-responsive homologues in the DtxR protein family.
Lieser
Scot A
SA
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0358, USA.
Davis
Talib C
TC
Helmann
John D
JD
Cohen
Seth M
SM
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001704:Biopolymers; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D001704:Biopolymers; D017931:DNA Primers; D004269:DNA, Bacterial; C433792:MntR protein, bacteria; D012097:Repressor Proteins