GLUTAMATE AT THE SITE OF PHOSPHORYLATION OF NITROGEN-REGULATORY PROTEIN NTRC MIMICS ASPARTYL-PHOSPHATE AND ACTIVATES THE PROTEIN
GLUTAMATE AT THE SITE OF PHOSPHORYLATION OF NITROGEN-REGULATORY PROTEIN NTRC MIMICS ASPARTYL-PHOSPHATE AND ACTIVATES THE PROTEIN
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DOI：
10.1006/jmbi.1993.1370
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发表时间：
1993-07-05
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：
KLOSE, KE;WEISS, DS;KUSTU, S
肠道细菌的NTRC蛋白是一种增强子结合蛋白，在氮限制条件下通过RNA聚合酶的σ54全酶形式激活转录，在体外NTRC必须被磷酸化以催化ATP水解并激活转录。鼠伤寒沙门氏菌NTRC的磷酸化位点是天冬氨酸54，位于蛋白质的氨基端调节结构域。我们用定点突变技术在第54位残基上进行“保守”的丙氨酸、天冬酰胺和谷氨酸的替换，并在体外和体内检测了突变体NTRC蛋白的性质，在体外，它们中的一个被磷酸化，正如预期的那样，如果D54实际上是唯一的磷酸化位点。D54 A和D54 N不激活glnA的转录，但有趣的是，D54 E组成性激活。与磷酸化野生型NTRC相比，D54 E的激活是部分的。将D54 A或D54 N与S160 F（NTRC中央结构域的变化，部分绕过磷酸化的要求）组合，产生了与单独携带S160 F的形式一样活性的双重突变蛋白，表明D54的变化不会对NTRC其余部分的功能产生不利影响。结合D54 E与S160 F增加了组成型ATP酶活性和转录激活的水平，高于单独携带单一变化的突变NTRC蛋白的水平。我们的结论是，天冬氨酸54磷酸化激活NTRC和假设D54E突变模拟磷酸化，从而允许NTRC水解ATP和激活转录。编码NTRC蛋白的突变株在D54处具有取代的表型表明，在不存在谷氨酰胺的情况下，NTRC在位置54处的磷酸化是正常生长所必需的，并且即使在不存在NTRB的情况下，这种磷酸化也在一定程度上发生。
The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the σ54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions.In vitroNTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription. The site of phosphorylation of NTRC fromSalmonella typhimuriumis Aspartate 54, which lies in the amino-terminal regulatory domain of the protein. We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteinsin vitroandin vivo.In vitronone of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation. D54A and D54N did not activate transcription ofglnAbut, interestingly, D54E activated constitutively. Activation by D54E was partial compared to that by phosphorylated wild-type NTRC. Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC. Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone. We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription. Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.