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首页> 外文期刊>Journal of bacteriology >The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli
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The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli

机译：氮同化控制蛋白（NAC）的氨基末端100个残基编码了产气克雷伯菌和大肠杆菌中NAC的所有已知特性

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The nitrogen assimilation control protein (NAC) fromKlebsiella aerogenes or Escherichia coli(NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within thenac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activatehut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACKfragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.
机译：产气克雷伯氏菌或大肠杆菌（NAC K 或NAC E ）的氮同化控制蛋白）是一种转录调节剂，它对于激活组氨酸利用（ hut ）操纵子的转录并抑制< em> K。气孔。测试了通过在 nac 开放阅读框中引入终止密码子而产生的截短的NAC多肽激活小屋和抑制 gdh 的能力。体内。大多数带有100个或更多氨基酸的NAC K 和NAC E 片段（野生型NAC K 和NAC E 都具有305个氨基酸）在体内激活 hut 和抑制 gdh 表达。全长NAC K 和NAC E 与麦芽糖结合蛋白（MBP）分离为嵌合蛋白。从这种嵌合体释放的NAC K 和NAC E 与NAC K一样，在体外纯化系统中能够激活 hut 转录。 129和NAC E 100（包含129个氨基酸的NAC K 片段和100个氨基酸的NAC E 片段）已发布来自类似的嵌合体。还生成了一组NAC E 和NAC K 片段，这些片段在其C末端带有结合镍的组氨酸标签（his 6 ）。源自NAC E 的所有此类构建体都是不溶的，NAC E 本身也是不溶的。在从NAC K 派生的带有 6 标签的构造中，NAC K 100处于非活动状态，而NAC K 120活跃。测试了几个NAC片段的二聚化。 NAC K 120-his 6 和NAC K 100-his 6 是溶液中的二聚体。 MBP-NAC K 和MBP-NAC K 129是溶液中的单体，但通过用Xa因子裂解释放MBP时会二聚。 MBP-NAC E 容易被因子Xa裂解，但所得的NAC E 也被蛋白酶降解。然而，MBP-NAC E -his6完全抵抗Xa因子的切割，表明该蛋白的C和N末端之间存在相互作用。

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