Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae.
Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae.
复制标题
氮同化控制蛋白形成四聚体对于强烈抑制肺炎克雷伯菌中谷氨酸脱氢酶形成的重要性。
DOI：
10.1128/jb.187.24.8291-8299.2005
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Bender,RobertA
中科院分区：
文献类型：
--
作者：
Rosario,ChristopherJ;Bender,RobertA
The nitrogen assimilation control protein (NAC) fromKlebsiella pneumoniaeis a very versatile regulatory protein. NAC activates transcription of operons such ashut(histidine utilization) andure(urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such asgdhA, whose products use ammonia. NAC exerts a weak repression atgdhAby competing with the binding of a lysine-sensitive activator. NAC also strongly represses transcription ofgdhA(about 20-fold) by binding to two separated sites, suggesting a model involving DNA looping. We have identified negative control mutants that are unable to exert this strong repression ofgdhAexpression but still activatehutandureexpression normally. Some of these negative control mutants (e.g., NAC86terand NAC132ter) delete the C-terminal domain, thought to be required for tetramerization. Other negative control mutants (e.g., NACL111Kand NACL125R) alter single amino acids involved in tetramerization. In this work we used gel filtration to show that NAC86terand NACL111Kare dimers in solution, even at high concentration (NACWTis a tetramer). Moreover, using a combination of DNase I footprints and gel mobility shifts assays, we showed that when NACWTbinds to two adjacent sites on a DNA fragment, NACWTbinds as a tetramer that bends the DNA fragment significantly. NACL111Kbinds to such a fragment as two independent dimers without inducing the strong bend. Thus, NACL111Kis a dimer in solution or when bound to DNA. NACL111K(typical of the negative control mutants) is wild type for every other property tested: (i) it activates transcription athutandure; (ii) it competes with the lysine-sensitive activator for binding atgdhA; (iii) it binds to the same sites at thehut,ure,nac, andgdhApromoters as NACWT; (iv) the relative affinity of NACL111Kfor these sites follows the same order as NACWT(ure>gdhA>nac>hut); (v) it induces the same slight bend as dimers of NACWT; and (vi) its DNase I footprints at these sites are indistinguishable from those of NACWT(except for features ascribed to tetramer formation). The only two phenotypes we know for negative control mutants of NAC are their inability to tetramerize and their inability to cause the strong repression ofgdhA. Thus, we propose that in order for NACWTto exert the strong repression, it must form a tetramer that bridges the two sites atgdhA(similar to other DNA looping models) and that the negative control mutants of NAC, which fail to tetramerize, cannot form this loop and thus fail to exert the strong repression atgdhA.
