σ54-dependent transcription activator phage shock protein F of Escherichia coli:: a fragmentation approach to identify sequences that contribute to self-association
σ54-dependent transcription activator phage shock protein F of Escherichia coli:: a fragmentation approach to identify sequences that contribute to self-association
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DOI：
10.1042/bj20031464
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发表时间：
2004-03-15
影响因子：
4.1
通讯作者：
Buck, M
中科院分区：
文献类型：
--
作者：
Bordes, P;Wigneshweraraj, SR;Buck, M
机械化学酶的AAA（与各种细胞活动相关的atp酶）超家族的蛋白质是多功能的，控制着广泛的细胞功能。许多AAA蛋白具有自结合成寡聚结构的共同特性，并利用核苷酸结合和水解来调节其生物输出。大肠杆菌转录激活因子PspF（噬菌体休克蛋白F）是AAA蛋白家族中sigma（54）依赖性转录激活因子的一员。核苷酸相互作用调节PspF的功能状态，使其能够自结合并与其靶标sigma(54)-RNAP （RNA聚合酶）闭合复合物相互作用。sigma（54）依赖性激活剂的AAA结构域内的自结合决定因素仍然缺乏表征。在本研究中，我们利用PspF的AAA结构域片段作为探针来研究PspF的核苷酸条件自结合。结果表明，PspF片段反式抑制PspF的特异性自结合。PspF片段阻止了核苷酸与PspF的有效结合，这与AAA蛋白寡聚物中核苷酸相互作用的位点在两个原聚体之间产生的观察结果一致。使用基于接近度的足迹和交联技术，我们证明了该片段中所代表的序列接近PspF低聚物中的一个原聚体-原聚体界面。由于该PspF片段中所代表的序列还包含一个与sigma(54)-RNAP闭合复合体相互作用的高度保守基序，我们认为PspF可能被组织为将核苷酸相互作用和自结合与sigma(54)-RNAP结合和转录激活联系起来。
Proteins that belong to the AAA (ATPases associated with various cellular activities) superfamily of mechanochemical enzymes are versatile and control a wide array of cellular functions. Many AAA proteins share the common property of self-association into oligomeric structures and use nucleotide binding and hydrolysis to regulate their biological output. The Escherichia coli transcription activator PspF (phage shock protein F) is a member of the sigma(54)-dependent transcriptional activators that belong to the AAA protein family. Nucleotide interactions condition the functional state of PspF, enabling it to self-associate and interact with its target, the sigma(54)-RNAP (RNA polymerase) closed complex. The self-association determinants within the AAA domain of sigma(54)- dependent activators remain poorly characterized. In the present study, we have used a fragment of the AAA domain of PspF as a probe to study the nucleotide-conditioned self-association of PspF. Results show that the PspF fragment acts in trans to inhibit specifically self-association of PspF. The PspF fragment prevented efficient binding of nucleotides to PspF, consistent with the observation that the site for nucleotide interactions within an oligomer of AAA proteins is created between two protomers. Using proximity-based footprinting and cross-linking techniques, we demonstrate that the sequences represented in this fragment are close to one protomer-protomer interface within a PspF oligomer. As the sequences represented in this PspF fragment also contain a highly conserved motif that interacts with the sigma(54)-RNAP closed complex, we suggest that PspF may be organized to link nucleotide interactions and self-association to sigma(54)-RNAP binding and transcription activation.