Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein
Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein
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DOI：
10.1128/jb.184.24.6777-6785.2002
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发表时间：
2002-12
影响因子：
3.2
通讯作者：
Francisca Reyes-Ramírez;R. Little;R. Dixon
中科院分区：
文献类型：
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作者：
Francisca Reyes-Ramírez;R. Little;R. Dixon
固氮菌vinelandii σ54依赖性转录激活蛋白NifA受NifL蛋白的调控，以响应氧化还原、碳和氮的状态。在不适合固氮的条件下，NifL通过形成NifL-NifA蛋白复合物抑制NifA的转录激活。NifL抑制NifA中心AAA+结构域的atp酶活性，该结构域是驱动σ54-RNA聚合酶形成开放复合物所必需的，也可能抑制激活剂-聚合酶的相互作用。为了更详细地分析抑制机制，我们分离了对NifL抑制作用具有抗性的NifA突变体。在NifA的氨基末端GAF结构域和催化AAA+结构域均分离出突变。在氮和氧化还原状态下，一些突变体阻断了NifL的抑制作用，而一些突变体NifL蛋白显然能够区分不同环境条件下存在的NifL形式。其中一个突变蛋白NifA-Y254N在厌氧氮过量条件下对NifL具有抗性，但在好氧生长条件下对NifL相对敏感。纯化的突变蛋白在体外的特性与体内表型一致，表明NifA-Y254N对NifL与A. vinelandii GlnK相互作用所传递的氮信号没有反应，但在ADP存在时对NifL的氧化形式有反应。我们的观察结果表明，NifL的不同构象可能在离散信号转导事件的响应中产生，并且NifL的GAF和AAA+结构域都参与了对NifL的响应。
ABSTRACT The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymerase interaction. To analyze the mechanism of inhibition in greater detail, we isolated NifA mutants which are resistant to the inhibitory action of NifL. Mutations in both the amino-terminal GAF domain and the catalytic AAA+ domain of NifA were isolated. Several mutants blocked inhibition by NifL in response to both nitrogen and redox status, whereas some of the mutant NifA proteins were apparently able to discriminate between the forms of NifL present under different environmental conditions. One mutant protein, NifA-Y254N, was resistant to NifL under conditions of anaerobic nitrogen excess but was relatively sensitive to NifL under aerobic growth conditions. The properties of the purified mutant protein in vitro were consistent with the in vivo phenotype and indicate that NifA-Y254N is not responsive to the nitrogen signal conveyed by the interaction of NifL with A. vinelandii GlnK but is responsive to the oxidized form of NifL when ADP is present. Our observations suggest that different conformers of NifL may be generated in response to discrete signal transduction events and that both the GAF and AAA+ domains of NifA are involved in the response to NifL.