Ghochikyan Anahit, Karaivanova Iovka Miltcheva, Lecocq Michèle, Vusio Patricia, Arnaud Marie-Claire, Snapyan Marina, Weigel Pierre, Guével Laetitia, Buckle Malcolm, Sakanyan Vehary
Laboratoire de Biotechnologie, FRE CNRS 2230, Unité Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, 44322 Nantes. IFR 26, INSERM, 44035 Nantes, France.
J Bacteriol. 2002 Dec;184(23):6602-14. doi: 10.1128/JB.184.23.6602-6614.2002.
Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the alpha4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the alpha4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.
嗜热脂肪芽孢杆菌的ArgR能高效结合大肠杆菌的carAB操纵基因，而大肠杆菌的阻遏蛋白与嗜热脂肪芽孢杆菌的argCo操纵基因的结合能力却很差。为了阐明ArgR之间这种相互矛盾的行为，我们通过交换N端DNA结合结构域和C端寡聚化结构域或交换连接肽构建了嵌合蛋白。携带大肠杆菌DNA结合结构域和嗜热脂肪芽孢杆菌寡聚化结构域的嵌合体与arg操纵基因表现出序列非特异性而非序列特异性相互作用。携带嗜热脂肪芽孢杆菌DNA结合结构域和大肠杆菌寡聚化结构域的嵌合体对嗜热脂肪芽孢杆菌的argCo和大肠杆菌的carAB操纵基因表现出高DNA结合亲和力，并在体外抑制了来自嗜热脂肪芽孢杆菌PargCo控制区的报告基因转录；精氨酸对它们的DNA结合亲和力没有影响，甚至使其降低。通过蛋白质阵列方法，我们发现野生型嗜热脂肪芽孢杆菌ArgR及其仅含有来自大肠杆菌ArgR交换连接肽或携带嗜热脂肪芽孢杆菌DNA结合结构域以及连接肽和α4区域的衍生物能够结合含有单个Arg框的argCo。这种结合比与双框操纵基因的结合弱，但不再依赖精氨酸。一系列观察结果表明，寡聚化结构域中的α4螺旋和连接肽有助于识别单个或双个Arg框，从而有助于来自两种远缘细菌的相似但不完全相同的ArgR阻遏蛋白对操纵基因DNA的结合特异性。
