Wu Y F, Datta P
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
J Bacteriol. 1992 Jan;174(1):233-40. doi: 10.1128/jb.174.1.233-240.1992.
A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.
大肠杆菌tdcABC操纵子启动子区域的一个14碱基对片段与大肠杆菌整合宿主因子（IHF）的共有结合位点具有序列同一性。在himA（IHF缺陷型）菌株中，tdcB'-'lacZ蛋白融合质粒表达的β-半乳糖苷酶约为同基因himA+菌株的10%。himA突变体中染色体tdcB基因的苏氨酸脱水酶活性也约为野生型酶水平的10%。在融合质粒中引入到假定的IHF结合位点的两种不同突变极大地降低了含IHF细胞中质粒编码的β-半乳糖苷酶活性。体外凝胶阻滞和DNase I足迹分析表明，纯化的IHF与野生型启动子结合，但不与突变型启动子结合。IHF保护了-118至-88之间一个包含保守的IHF共有序列的31碱基对区域。这些结果表明，tdc操纵子在体内的有效表达需要功能性的IHF和tdc启动子中的IHF结合位点。
