Li J, Kustu S, Stewart V
Section of Microbiology, Cornell University, Ithaca, NY 14853.
J Mol Biol. 1994 Aug 12;241(2):150-65. doi: 10.1006/jmbi.1994.1485.
The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression. Mutational studies and sequence comparisons have suggested that NarL protein binding sites contain heptameric sequences related to the consensus, TACYNMT (where Y = C or T, M = A or C, and N = any nucleotide). There are four NarL heptamers in the -105 region of the fdnGHI (formate dehydrogenase-N) operon, and mutational analysis supports the role of these heptamers in nitrate induction. To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL). A constitutive mutant form with a single substitution (V88A) in the amino-terminal (response regulator) region was used. The MBP-NarL (V88A) protein protected all four heptamers in the fdnG operon control region from DNase I cleavage. Identical footprints were observed with NarL (V88A) protein that had been proteolytically cleaved free from the MBP domain. Binding of MBP-NarL (V88A) protein to the four heptamers in the -105 region of the fdnG operon appeared to be cooperative, and occupancy of the central heptamers was necessary for occupancy of the flanking heptamers. In addition to the V88A substitution, a low molecular weight phosphodonor, such as acetyl phosphate, was required for observable footprints. This indicates that phosphorylation of the NarL protein enhances its affinity for its multiple DNA targets in the fdnG operon, perhaps by increasing protein-protein interactions rather than protein-DNA interactions. We also performed footprinting studies at the narGHJI (nitrate reductase), narK (nitrite efflux), and frdABCD (fumarate reductase) operon control regions. Extensive areas of each control region were protected from DNase I attack by phosphorylated MBP-NarL (V88A) protein. The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -200. Mutational analysis indicates that the NarL heptamer centered at position -89, in addition to the previously-identified -200 region, is involved in nitrate induction. Comparisons of the four operon control regions studied indicate that the NarL heptamers are arranged with diverse orientations and spacing.
narL基因产物是一种对硝酸盐有反应的厌氧呼吸基因表达激活剂和阻遏物。突变研究和序列比较表明，NarL蛋白结合位点包含与共有序列TACYNMT（其中Y = C或T，M = A或C，N = 任何核苷酸）相关的七聚体序列。在甲酸脱氢酶-N（fdnGHI）操纵子的-105区域有四个NarL七聚体，突变分析支持这些七聚体在硝酸盐诱导中的作用。为了研究NarL与DNA的相互作用，我们将NarL蛋白纯化成为麦芽糖结合蛋白（MBP）融合蛋白（MBP-NarL）。使用了在氨基末端（反应调节子）区域有单个取代（V88A）的组成型突变体形式。MBP-NarL（V88A）蛋白保护fdnG操纵子控制区域中的所有四个七聚体免受DNase I切割。从MBP结构域蛋白水解切割下来的NarL（V88A）蛋白也观察到相同的足迹。MBP-NarL（V88A）蛋白与fdnG操纵子-105区域中的四个七聚体的结合似乎是协同的，并且占据中央七聚体对于侧翼七聚体的占据是必要的。除了V88A取代外，还需要低分子量的磷酸供体，如乙酰磷酸，才能观察到足迹。这表明NarL蛋白的磷酸化增强了其对fdnG操纵子中多个DNA靶点的亲和力，可能是通过增加蛋白质-蛋白质相互作用而不是蛋白质-DNA相互作用。我们还在硝酸盐还原酶（narGHJI）、亚硝酸盐外排（narK）和延胡索酸还原酶（frdABCD）操纵子控制区域进行了足迹研究。磷酸化的MBP-NarL（V88A）蛋白保护每个控制区域的大片区域免受DNase I攻击。narG操纵子控制区域从-50到-110位置受到保护，并且在较高蛋白质浓度下，在-200位置附近也受到保护。突变分析表明，除了先前确定的-200区域外，位于-89位置的NarL七聚体也参与硝酸盐诱导。对所研究的四个操纵子控制区域的比较表明，NarL七聚体以不同的方向和间距排列。
