The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions.
The Escherichia coli NarL receiver domain regulates transcription through promoter specific functions.
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DOI：
10.1186/s12866-015-0502-9
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发表时间：
2015-08-26
期刊：
影响因子：
4.2
通讯作者：
Gunsalus RP
中科院分区：
文献类型：
--
作者：
Katsir G;Jarvis M;Phillips M;Ma Z;Gunsalus RP
大肠杆菌反应调节因子NarL控制厌氧症期间参与硝酸盐呼吸的基因转录。NarL由两个域组成，由一个围绕域间接口的链接器连接。NarL n端受体结构域（RD）的磷酸化释放了原本被隔离的c端输出结构域（OD），该结构域随后结合特定的DNA启动子位点，抑制或激活基因表达。本研究的目的是探讨NarL OD和RD在多大程度上独立调节转录，以及连接物对OD功能的影响。我们检测了含有不同连接子片段的NarL OD构建体抑制frdA-lacZ或激活narG-lacZ报告融合基因的能力。这些体内表达分析表明，无论是否存在连接体螺旋α6， NarL OD都能组成性地抑制frdA-lacZ的表达，而不考虑硝酸盐的可用性。然而，连接环α5-α6的存在逆转了这种抑制，并且在体外也显示出DNA结合受损。单纯OD不能激活narG-lacZ的表达；这种活性需要NarL RD的存在。足迹分析表明，NarL OD仅部分结合narG启动子上的识别位点，并且磷酸化的RD的存在增加了结合亲和力。用于检查结构域寡聚化的分析超浓缩表明，NarL RD在溶液中形成二聚体，而OD是单体。NarL RD作为一个开关，以硝酸盐响应的方式关闭或释放OD，但还有其他作用，直接刺激OD缺乏独立功能的启动子的转录。RD的一个作用是增强OD与目标启动子位点的DNA结合亲和力。数据还表明，NarL磷酸化导致RD二聚化和整个连接区域从OD分离。本文的在线版本（doi:10.1186/s12866-015-0502-9）包含补充材料，可供授权用户使用。
The Escherichia coli response regulator NarL controls transcription of genes involved in nitrate respiration during anaerobiosis. NarL consists of two domains joined by a linker that wraps around the interdomain interface. Phosphorylation of the NarL N-terminal receiver domain (RD) releases the, otherwise sequestered, C-terminal output domain (OD) that subsequently binds specific DNA promoter sites to repress or activate gene expression. The aim of this study is to investigate the extent to which the NarL OD and RD function independently to regulate transcription, and the affect of the linker on OD function. NarL OD constructs containing different linker segments were examined for their ability to repress frdA-lacZ or activate narG-lacZ reporter fusion genes. These in vivo expression assays revealed that the NarL OD, in the absence or presence of linker helix α6, constitutively repressed frdA-lacZ expression regardless of nitrate availability. However, the presence of the linker loop α5-α6 reversed this repression and also showed impaired DNA binding in vitro. The OD alone could not activate narG-lacZ expression; this activity required the presence of the NarL RD. A footprint assay demonstrated that the NarL OD only partially bound recognition sites at the narG promoter, and the binding affinity was increased by the presence of the phosphorylated RD. Analytical ultracentrifugation used to examine domain oligomerization showed that the NarL RD forms dimers in solution while the OD is monomeric. The NarL RD operates as an on-off switch to occlude or release the OD in a nitrate-responsive manner, but has additional roles to directly stimulate transcription at promoters for which the OD lacks independent function. One such role of the RD is to enhance the DNA binding affinity of the OD to target promoter sites. The data also imply that NarL phosphorylation results in RD dimerization and in the separation of the entire linker region from the OD. The online version of this article (doi:10.1186/s12866-015-0502-9) contains supplementary material, which is available to authorized users.