Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding
Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding
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DOI：
10.1128/jb.01550-06
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发表时间：
2007-02-01
影响因子：
3.2
通讯作者：
Taguchi, Seiichi
中科院分区：
文献类型：
--
作者：
Yamada, Miwa;Yamashita, Koichi;Taguchi, Seiichi
来自嗜热副球菌的PhaR作为编码相蛋白（PhaP）和PhaR本身的基因表达的阻遏物或自动调节物起作用，相蛋白和PhaR两者都是聚羟基链烷酸酯（PHA）颗粒的组分（A. Maehara，S.田口，T.西山T. Yamane和Y. Doi，J. Bacteriol. 184：3992-4002，2002）。PhaR是一种独特的调节蛋白，因为它也具有与效应分子PRA聚酯紧密结合的能力。在本研究中，通过使用石英晶体微量天平，我们获得了PhaR同时与靶DNA和聚[（R）-3-羟基丁酸酯] [P（3 HB）]（PHAs之一）结合的直接证据。为了鉴定负责DNA结合的PhaR氨基酸残基，用编码PhaR蛋白的基因进行缺失和PCR介导的随机点突变实验。通过体内监测检测系统结合大肠杆菌中绿色荧光蛋白的基因表达，有效地筛选了DNA结合能力降低的PhaR点突变体。在大肠杆菌中表达的PhaR野生型和突变体的DNA结合能力。使用凝胶位移测定和表面等离子体共振分析评估大肠杆菌。这些实验表明，碱性氨基酸和酪氨酸的N-末端区域，这是高度保守的PhaR同系物，负责DNA结合。然而，大多数DNA结合能力降低的突变体与P（3 HB）结合的能力不受影响，这强烈表明PhaR具有能够与靶DNA和P（3 HB）结合的两个独立的结构域。
PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PRA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).