Marincs Ferenc, Manfield Iain W, Stead Jonathan A, McDowall Kenneth J, Stockley Peter G
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Biochem J. 2006 Jun 1;396(2):227-34. doi: 10.1042/BJ20060021.
We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.
我们利用DNA阵列研究了敲除甲硫氨酸阻遏基因metJ对大肠杆菌转录组的影响。我们检测了在敲除菌株中从表达质粒提供野生型或突变型MetJ阻遏物的效果，从而建立了一种快速检测方法，用于检测先前在体外鉴定的突变在体内的影响。阻遏作用主要局限于参与甲硫氨酸生物合成和摄取的已知基因。然而，我们鉴定出了一些在没有阻遏物时显著上调的其他基因。对这些基因5'启动子区域的序列分析确定了其中三个基因与met-box序列有合理匹配，随后的电泳迁移率变动分析表明，对于其中两个这样的位点，它们的阻遏物亲和力高于或与已知的metB操纵子相当，这表明它们受到直接调控。对于其中一个位点folE，通过其编码酶的代谢作用可以对此进行合理解释；然而，与其他受调控位点的联系尚不清楚，这表明已知的met调控子有所扩展，并且阻遏物的作用更加复杂。质粒基因替换系统已被用于利用结构特征明确的突变阻遏物Q44K研究蛋白质-蛋白质协同作用在操纵子饱和中的重要性。在体内，观察到的调控水平有可检测到的降低，这证明了平衡蛋白质-蛋白质和蛋白质-DNA亲和力的重要性。
