NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro
NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro
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DOI：
10.1006/jmbi.1998.2105
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发表时间：
1998-10-23
影响因子：
5.6
通讯作者：
Stewart, V
中科院分区：
文献类型：
--
作者：
Chai, WH;Stewart, V
在肺炎克雷伯菌中，硝酸盐同化所需的酶是由nasFEDCBA操纵子编码的。以往的遗传学研究得出结论，硝酸盐和亚硝酸盐诱导NASF操纵子的表达是由转录抗终止机制决定的。在亚硝酸盐存在的情况下，假设Nasr基因产物抑制位于NASF操纵子前导区的因子非依赖终止子位置的转录终止。为了在体外测试这个模型，我们首先将Nasr纯化为麦芽糖结合蛋白融合形式(MBP-Nasr)和组氨酸(His(6))标记形式(His(6)-Nasr)。体外转录模板含有NASF操纵子前导区，用依赖于sigma(70)的tac启动子取代依赖于sigma(N)的启动子。我们发现，在体外，前导模板的转录终止于终止子位点，MBP-Nasr和His(6)-Nasr蛋白都能引起该位点的转录通读，以响应硝酸盐或亚硝酸盐。半最大抗终止需要中等浓度的硝酸盐或亚硝酸盐(1-10µM)，其他几种阴离子，包括氯酸盐，都没有作用。先前对前导缺失的体内分析确定了负调控(终止子)和正调控所需的区域。这些缺失模板的体外转录结果与体内分析完全相关。最后，凝胶迁移率漂移分析表明，His(6)-Nasr与NASF前导RNA特异结合。这种结合在体外是独立于硝酸盐的。这些结果有力地支持了先前体内分析得出的结论，并建立了Nasr通过与NASF领导RNA相互作用来介导配体反应性转录抗终止的结论。(C)1998年学术出版社。
In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. in the presence of nitrate of nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His(6)-tagged form (His(6)-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma(70)-dependent tac promoter for the native sigma(N)-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His(6)-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 mu M) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified regions required for both negative regulation (the terminator) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. Finally, electrophoresis mobility shift analysis revealed that His(6)-NasR bound specifically to nasF leader RNA. This binding was independent of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction with nasF leader RNA. (C) 1998 Academic Press.