Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter
Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter
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DOI：
10.1046/j.1365-2958.2001.02518.x
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发表时间：
2001-07-01
影响因子：
3.6
通讯作者：
Skorupski, K
中科院分区：
文献类型：
--
作者：
Kovacikova, G;Skorupski, K
霍乱弧菌毒力因子、毒素共调节菌毛（TCP）和霍乱毒素（CT）的表达依赖于LysR调节剂AphB与第二种蛋白AphA合作以激活膜结合转录因子TcpP和TcpH表达的能力。为了深入了解AphA和AphB合作激活tcpPH表达的机制，我们已经将这两种蛋白质纯化到接近同质性，并表明它们各自能够在不同的结合位点与经典的tcpPH启动子相互作用。如通过tcpP-lacZ启动子缺失实验、凝胶位移和DNA酶I足迹法所示，AphA结合到启动子的从转录开始的-101和-71之间的区域并从该区域激活。AphB结合到-78和-43之间的部分重叠下游位点并从其激活，并且这些功能依赖于类似于充分表征的LysR结合基序的部分二联体对称区域。先前已显示在二分体对称性的该区域中的单个碱基对差异在霍乱弧菌的两种致病生物型（经典型和El Tor型）之间的毒力基因表达中起关键作用。我们还发现tcpPH启动子受到全局调节因子cAMP-CRP的负面影响。纯化的CRP以cAMP依赖性方式与tcpPH启动子中的近共有序列结合，并保护完全在AphA和AphB保护区域内的区域免受DNA酶I消化。这些发现提出了这样的可能性，即cAMP-CRP对毒力基因表达的负面影响是其在各种条件下影响AphA和AphB依赖的tcpPH转录激活的能力的结果。
The expression of the Vibrio cholerae virulence factors, toxin-co-regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co-operate with a second protein, AphA, to activate the expression of the membrane-bound transcription factors TcpP and TcpH. To gain insights into the mechanism by which AphA and AphB co-operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites. As shown by tcpP-lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between -101 and -71 from the start of transcription. AphB binds to and activates from a partially overlapping downstream site between -78 and -43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well-characterized LysR-binding motif. A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease-causing biotypes of V. cholerae, classical and El Tor. We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP-CRP. Purified CRP binds to a near-consensus sequence in the tcpPH promoter in a cAMP-dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB. These findings raise the possibility that the negative effect of cAMP-CRP on virulence gene expression is the result of its ability to influence AphA- and AphB-dependent transcriptional activation of tcpPH under various conditions.