Antognoni F, Del Duca S, Kuraishi A, Kawabe E, Fukuchi-Shimogori T, Kashiwagi K, Igarashi K
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
J Biol Chem. 1999 Jan 22;274(4):1942-8. doi: 10.1074/jbc.274.4.1942.
Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.
利用由potA、-B、-C和-D基因组成的亚精胺摄取操纵子，对大肠杆菌中亚精胺摄取的抑制作用进行了研究，这种抑制作用发生在多胺积累的情况下。PotD是一种通常存在于周质中的亚精胺结合蛋白，它抑制了potABCD操纵子的转录，并且亚精胺增强了PotD的抑制作用。转录不受牛血清白蛋白、PotA或PotF的影响，这表明PotD的作用对PotD蛋白具有特异性。在存在8 mM亚精胺的情况下，当模板DNA与PotD的摩尔比约为1:500时，观察到转录受到50%的抑制。发现PotD与potA基因ATG起始密码子上游-258至-209核苷酸区域以及下游+66至+135核苷酸区域结合。亚精胺刺激PotD与下游位点的结合。在大肠杆菌DH5α中过表达PotD会抑制亚精胺的摄取、PotABCD mRNA的合成以及与含有PotD结合位点的potA基因下游融合的lacZ报告基因的表达。在过表达PotD的细胞中，大量的PotD以PotD前体的形式存在于原生质球中。我们的结果表明，PotD前体也可以抑制亚精胺转运。使用突变的PotD蛋白确定了PotD中与其与potABCD操纵子相互作用有关的氨基酸残基。发现PotD的Thr-35和Ser-85对于这种相互作用很重要。这些结果表明，亚精胺转运（potABCD）操纵子的转录在体内受到PotD前体而非PotD的抑制，PotD前体通过与操纵子转录起始位点附近的两个区域结合来实现抑制作用。
