CONSTITUTIVE FORMS OF THE ENHANCER-BINDING PROTEIN NTRC - EVIDENCE THAT ESSENTIAL OLIGOMERIZATION DETERMINANTS LIE IN THE CENTRAL ACTIVATION DOMAIN
CONSTITUTIVE FORMS OF THE ENHANCER-BINDING PROTEIN NTRC - EVIDENCE THAT ESSENTIAL OLIGOMERIZATION DETERMINANTS LIE IN THE CENTRAL ACTIVATION DOMAIN
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DOI：
10.1006/jmbi.1995.0330
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发表时间：
1995-06-16
影响因子：
5.6
通讯作者：
KUSTU, S
中科院分区：
文献类型：
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作者：
FLASHNER, Y;WEISS, DS;KUSTU, S
氮调节蛋白C(NTRC)是一种细菌增强子结合蛋白，通过Sigma(54)-全酶激活转录。为了激活转录，NTRC必须水解三磷酸腺苷，这一反应依赖于它被磷酸化并形成适当的寡聚体。在这篇文章中，我们描述了鼠伤寒沙门氏菌NTRC蛋白的“构成”突变形式；与野生型NTRC不同，这些突变形式能够在体外不被磷酸化的情况下水解三磷酸腺苷并激活转录。NTRC(构成)蛋白中改变的氨基酸位于N-末端调节域和中央结构域，后者直接负责转录激活。被改变的残基在sigma(54)-全酶的激活剂中不是保守的，甚至在来自蛋白质细菌(紫色细菌)不同亚群成员的NT C蛋白中也不相同。正常情况下，NTRC(构成蛋白)被磷酸化；磷酸化增强了它们水解三磷酸腺苷和激活转录的能力。此外，当这些蛋白质与增强子结合时发生的寡聚化也增加了未修饰和磷酸化形式的ATPase活性。从两个在中央结构域有氨基酸取代的NTRC(构成蛋白)(NTRC(S160F)和NTRC(V2881))上移除N-末端调节域使它们保持活性，表明必要的寡聚决定因素存在于调节域之外。当Delta N-NTRC(S160F)与增强子结合时，它的ATPase活性被极大地刺激，并且该蛋白与一种不能与DNA结合的NTRC形式协同激活转录的能力证实了这一结论。再加上先前的结果表明寡聚决定簇不位于NTRC的C-末端DNA结合域；这些结果提供了它们位于中心区域的证据。
Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma(54)-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize ''constitutive'' mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrC(constitutive) proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma(54)-holoenzyme, and are not identical even among Nt C proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrC(constitutive) proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms.Removal of the N-terminal regulatory domain from two NtrC(constitutive) proteins with amino acid substitutions in the central domain (NtrC(S160F) and NtrC(V2881)) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of Delta N-NtrC(S160F) is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.