Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora
Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora
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DOI：
10.1007/s004380050547
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发表时间：
1997-09-01
期刊：
影响因子：
--
通讯作者：
Bernhard, F
中科院分区：
文献类型：
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作者：
Kelm, O;Kiecker, C;Bernhard, F
在大肠杆菌中表达了梨火疫病菌和大肠杆菌的RcsA和RcsB蛋白。coli中进行纯化。它们的DNA结合活性进行了检查，使用1-kb的DNA区域含有推定的启动子的ams操纵子的Ew。amylovora，其负责胞外多糖amylovoran的生物合成。迁移率变动分析表明RcsA和RcsB特异性结合到相对于操纵子的第一开放阅读框的翻译起始跨越核苷酸位置-578至-501的78 bp区域。该区域包括与E. coli sigma（70）启动子共有序列，并与E. coli cps启动子区。在JUMPstart共识中没有发现Rcs蛋白的结合，这对于多糖基因簇的各种启动子是典型的。单独的RcsA没有检测到DNA结合活性，只有高浓度的RcsB能够与ams启动子相互作用。这两种蛋白质在ams启动子的指定区域协同结合，并提供了进一步的证据，表明形成的DNA-蛋白质复合物涉及RcsA和RcsB的异源二聚体。在大肠杆菌中表达后，RcsA的比活性增强，而RcsB的比活性没有增强。相对于37 ℃的表达，此外，DNA-蛋白质复合物的形成受温度的影响。急诊coli RcsA/RcsB蛋白与ams启动子的相同区域结合，并且能够与来自Ew.食淀粉的。
The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions -578 to -501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coli sigma(70) promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28 degrees C, relative to expression at 37 degrees C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Res proteins from Ew. amylovora.