The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain.
The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain.
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肠道细菌氮调节蛋白 NTRC 二聚化的主要决定因素在于其羧基末端结构域。
DOI：
10.1006/jmbi.1994.1492
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发表时间：
1994
影响因子：
5.6
通讯作者：
Kustu,S
中科院分区：
文献类型：
--
作者：
Klose,KE;North,AK;Stedman,KM;Kustu,S
肠道细菌的 NTRC 蛋白（氮调节蛋白 C）是一种增强子结合蛋白，可通过 RNA 聚合酶的 σ54-全酶形式激活转录。 NTRC 是一种同二聚体蛋白，可结合 DNA 中的二元对称位点。为了激活转录，NTRC 必须被磷酸化，并且必须在增强子处形成适当的寡聚体。为了研究 NTRC 二聚体之间的亚基交换，我们构建了麦芽糖结合蛋白 (MBP) 与 NTRC 氨基末端的融合体 (MBP-NTRC)，并通过 DNA 结合的凝胶迁移率变化测定。当 MBP-NTRC 与野生型 NTRC 在 37°C 混合时，亚基交换迅速发生。 NTRC 同型二聚体解离的表观半衰期在 37°C 下为 2 至 3 分钟，并且不会因磷酸化而改变。分离的 NTRC 羧基末端结构域（91 个氨基酸残基）与野生型 NTRC 和 MBP-NTRC 形成异二聚体，表明 C 末端结构域足以进行二聚化。 C端结构域同二聚体的表观解离速率与全长NTRC基本相同，表明该蛋白的主要二聚化决定因素在于其C端结构域。与此一致，去掉最后 58 个氨基酸残基的 NTRC 的截短形式是溶液中的单体。此外，除去最后16或26个氨基酸残基的截短形式的NTRC在溶液中主要是单体，其C端结构域中具有氨基酸取代A410E的突变形式也是如此。上述NTRC突变形式的单体化可以基于NTRC的C端区域和用于反转刺激的因子的50个氨基酸残基区域之间的同源性合理化 (FIS) 蛋白质。
The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the σ54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer.In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRC at 37°C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37°C and is not changed by phosphorylation.The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTRC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.