Grunden A M, Self W T, Villain M, Blalock J E, Shanmugam K T
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, USA.
J Biol Chem. 1999 Aug 20;274(34):24308-15. doi: 10.1074/jbc.274.34.24308.
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.
编码钼酸盐特异性转运蛋白的大肠杆菌modABCD操纵子在体内以钼酸盐依赖的方式被ModE抑制。体外DNase I足迹实验确定了ModE-钼酸盐在modA操纵子/启动子DNA上的三个不同保护区域，即GTTATATT（-15至-8；区域1）、GCCTACAT（-4至+4；区域2）和GTTACAT（+8至+14；区域3）。在受保护DNA的这三个区域内，可以识别出一个五聚体序列TAYAT（Y = C或T）。DNA电泳迁移率实验表明，受保护区域1和2对于ModE-钼酸盐与DNA的结合至关重要，而受保护区域3增加了DNA与阻遏物的亲和力。发现这种相互作用的化学计量比为每个modA操纵子DNA有两个ModE-钼酸盐。5 nM的ModE-钼酸盐完全保护modABCD操纵子/启动子DNA免受DNase I催化的水解，而单独的ModE即使在100 nM时也无法保护DNA。modA操纵子DNA与ModE-钼酸盐之间相互作用的表观解离常数（K(d)）为0.3 nM，在没有钼酸盐的情况下K(d)增加到8 nM。在测试的各种含氧阴离子中，只有钨酸盐在ModE对modA的抑制中取代了钼酸盐，但ModE-钨酸盐对modABCD操纵子DNA的亲和力比对ModE-钼酸盐的亲和力低6倍。一种即使在没有钼酸盐的情况下也能抑制modA-lac的突变型ModE(T125I)蛋白，在没有钼酸盐的情况下保护modA操纵子DNA的相同区域。在有钼酸盐存在时，modA操纵子DNA与ModE(T125I)之间相互作用的表观K(d)为3 nM，在没有钼酸盐时为4 nM。钼酸盐与ModE的结合导致荧光发射减少，表明钼酸盐结合后蛋白质发生了构象变化。突变型ModE蛋白ModE(T125I)和ModE(Q216*)的荧光发射光谱不受钼酸盐影响。不依赖钼酸盐的突变型ModE蛋白在构象上显然模拟了天然的ModE-钼酸盐复合物，该复合物与TATAT-7bp-TAYAT DNA序列基序结合。
