CHIMERIC TRANSCRIPTIONAL ACTIVATORS GENERATED IN-VIVO FROM VNFA AND ANFA OF AZOTOBACTER-VINELANDII - N-TERMINAL DOMAIN OF ANFA IS RESPONSIBLE FOR DEPENDENCE ON NITROGENASE FE PROTEIN
CHIMERIC TRANSCRIPTIONAL ACTIVATORS GENERATED IN-VIVO FROM VNFA AND ANFA OF AZOTOBACTER-VINELANDII - N-TERMINAL DOMAIN OF ANFA IS RESPONSIBLE FOR DEPENDENCE ON NITROGENASE FE PROTEIN
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DOI：
10.1128/jb.176.21.6545-6549.1994
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发表时间：
1994-11-01
影响因子：
3.2
通讯作者：
DRUMMOND, M
中科院分区：
文献类型：
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作者：
FRISE, E;GREEN, A;DRUMMOND, M
通过串联克隆其结构基因并选择插入它们之间的条件致死基因，构建了在 Azotobacter vinelandii 的转录激活子 VnfA 和 AnfA 之间产生嵌合体的体内重组体。亲本分子的启动子特异性不同，AnfA（而非 VnfA）需要固氮酶的 Fe 蛋白才能发挥其活性。发现中央结构域 N 端一半具有融合连接的嵌合体没有活性，可能是错误折叠的结果。所有携带AnfA C端结构域的嵌合体都显示出相应的启动子特异性，支持将启动子特异性归因于C端结构域的DNA结合特性的模型。没有一个嵌合体表现出对AnfA典型的Fe蛋白的依赖性，包括由82%的AnfA组成且仅在N末端有一小段VnfA的嵌合体。删除 AnfA 的 N 端结构域得到完全活性的蛋白质，该蛋白质也独立于 Fe 蛋白质。这表明 N 末端结构域对活性具有抑制作用，而 Fe 蛋白可以缓解这种抑制作用。
In vivo recombinants generating chimeras between the transcriptional activators VnfA and AnfA of Azotobacter vinelandii were constructed by cloning their structural genes in tandem and selecting against a conditionally lethal gene inserted between them. The parent molecules differ in their promoter specificities and in that AnfA, but not VnfA, requires the Fe protein of nitrogenase for its activity. Chimeras with fusion junctions in the N-terminal half of the central domain were found to be inactive, probably as a result of misfolding. Ail chimeras carrying the C-terminal domain of AnfA showed the corresponding promoter specificity, supporting the model which ascribes promoter specificity to the DNA-binding properties of the C-terminal domain. None of the chimeras showed the dependence on Fe protein typical of AnfA, including one which composed 82% of AnfA with only a short segment of VnfA at the N terminus. Deleting the N-terminal domain of AnfA gave a fully active protein which was also independent of Fe protein. This indicates that the N-terminal domain has an inhibitory effect on activity which is relieved by Fe protein.