Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis
Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis
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DOI：
10.1128/jb.187.2.512-521.2005
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Kok, J
中科院分区：
文献类型：
--
作者：
den Hengst, CD;Curley, P;Kok, J
乳酸乳球菌MG 1363的CodY是抑制蛋白水解系统的几个编码蛋白的基因的表达的转录调节因子。这些基因包括pepN、pepC、opp-pepO 1，可能还有prtPM、pepX和pepDA 2，因为后三个基因相对于氮利用率的表达与前者相似。通过体外DNA结合试验和DNA酶I足迹技术，我们证明了L。lactis CodY直接与迄今为止已知的其主要靶标（opp系统）的启动子上游的区域相互作用。我们的研究结果表明，多个分子的CodY相互作用与此启动子和结合的CodY分子的量是受支链氨基酸的存在下，而不是由GTP。添加这些氨基酸强烈影响DNA酶I足迹中CodY保护区域的范围。oppD的上游区域的随机和定点诱变产生的变体在具有过量氮源的培养基中被去阻遏。结合研究揭示了CodY识别所需的启动子区域中特定碱基的重要性。
CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.