An analysis of the binding of repressor protein ModE to modABCD (molybdate transport) operator/promoter DNA of Escherichia coli.
Expression of the modABCD operon in Escherichia coli, which codes for a molybdate-specific transporter, is repressed by ModE in vivo in a molybdate-dependent fashion. In vitro DNase I-footprinting experiments identified three distinct regions of protection by ModE-molybdate on the modA operator/promoter DNA, GTTATATT (-15 to -8; region 1), GCCTACAT (-4 to +4; region 2), and GTTACAT (+8 to +14; region 3). Within the three regions of the protected DNA, a pentamer sequence, TAYAT (Y = C or T), can be identified. DNA-electrophoretic mobility experiments showed that the protected regions 1 and 2 are essential for binding of ModE-molybdate to DNA, whereas the protected region 3 increases the affinity of the DNA to the repressor. The stoichiometry of this interaction was found to be two ModE-molybdate per modA operator DNA. ModE-molybdate at 5 nM completely protected the modABCD operator/promoter DNA from DNase I-catalyzed hydrolysis, whereas ModE alone failed to protect the DNA even at 100 nM. The apparent K(d) for the interaction between the modA operator DNA and ModE-molybdate was 0.3 nM, and the K(d) increased to 8 nM in the absence of molybdate. Among the various oxyanions tested, only tungstate replaced molybdate in the repression of modA by ModE, but the affinity of ModE-tungstate for modABCD operator DNA was 6 times lower than with ModE-molybdate. A mutant ModE(T125I) protein, which repressed modA-lac even in the absence of molybdate, protected the same region of modA operator DNA in the absence of molybdate. The apparent K(d) for the interaction between modA operator DNA and ModE(T125I) was 3 nM in the presence of molybdate and 4 nM without molybdate. The binding of molybdate to ModE resulted in a decrease in fluorescence emission, indicating a conformational change of the protein upon molybdate binding. The fluorescence emission spectra of mutant ModE proteins, ModE(T125I) and ModE(Q216*), were unaffected by molybdate. The molybdate-independent mutant ModE proteins apparently mimic in its conformation the native ModE-molybdate complex, which binds to a DNA sequence motif of TATAT-7bp-TAYAT.
Grunden
A M
AM
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, USA.
Self
W T
WT
Villain
M
M
Blalock
J E
JE
Shanmugam
K T
KT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism*; D009876:Operon; D033902:Periplasmic Binding Proteins; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C098002:ModA protein, E coli; C491064:ModE protein, E coli; C101050:ModE protein, bacteria; D033902:Periplasmic Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C044659:molybdate; D008982:Molybdenum