Ohnishi K, Matsubara K, Hattori Y, Sadanari H, Yamada R, Fukuda S
Department of Microbiology, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan.
Biochim Biophys Acta. 1999 May 14;1445(2):196-206. doi: 10.1016/s0167-4781(99)00043-3.
brnQ is the gene encoding the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium. The expression of the gene is transcriptionally repressed by an excess of glycyl-l-leucine added to the bacterial culture. To investigate the mechanism of regulation, we constructed brnQ-lacZ translational fusions with various deletions upstream from the promoter of brnQ, and examined the effects of the deletions on the regulation. We found a cis-acting region, 5'-GTGTTTTA-3', for the repression of brnQ expression, which was located 94 base pairs upstream from the transcription start site. Removal of the sequence resulted in derepression of brnQ. Two homologous sequences were found 45 base pairs downstream and 42 base pairs upstream from the sequence. We designated these sequences as O1, O2, and O3, in the order from the sequence proximal to the promoter to that distal to the promoter, respectively. The gleR1 mutation, which we reported previously to be a regulatory mutation enhancing transcription of brnQ, was a G-to-T transversion in the O1 sequence 50 base pairs upstream from the transcription start site. Insertion of five nucleotides between O1 and O2 resulted in derepression of brnQ. Further insertion of five nucleotides did not restore the original regulation of brnQ, indicating the importance of the proper spacing of these sequences. We also showed that the protein product of livS, the gene responsible for regulation of the LIV-I transport system, may bind to the O2 sequence. Furthermore, LivS was shown to be an allele of Lrp based on complementation experiments.
brnQ是编码鼠伤寒沙门氏菌中支链氨基酸LIV-II转运系统的基因。该基因的表达受到添加到细菌培养物中的过量甘氨酰-L-亮氨酸的转录抑制。为了研究调控机制，我们构建了brnQ-lacZ翻译融合体，其在brnQ启动子上游有各种缺失，并研究了这些缺失对调控的影响。我们发现了一个顺式作用区域5'-GTGTTTTA-3'，用于抑制brnQ的表达，该区域位于转录起始位点上游94个碱基对处。去除该序列导致brnQ的去抑制。在该序列下游45个碱基对和上游42个碱基对处发现了两个同源序列。我们分别将这些序列从靠近启动子的序列到远离启动子的序列依次命名为O1、O2和O3。我们之前报道的gleR1突变是一个增强brnQ转录的调控突变，它是转录起始位点上游50个碱基对处O1序列中的一个G到T的颠换。在O1和O2之间插入五个核苷酸导致brnQ的去抑制。进一步插入五个核苷酸并没有恢复brnQ的原始调控，这表明这些序列的适当间距很重要。我们还表明，负责LIV-I转运系统调控的livS基因的蛋白质产物可能与O2序列结合。此外，基于互补实验，LivS被证明是Lrp的一个等位基因。
