Weissenborn D L, Wittekindt N, Larson T J
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
J Biol Chem. 1992 Mar 25;267(9):6122-31.
The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.
glpFK操纵子在大肠杆菌K-12的连锁图谱上位于约88分钟处，glpF启动子位于近端。glpF基因编码一种细胞质膜蛋白，可促进甘油扩散进入细胞。glpK基因编码甘油激酶。在本研究中，测定了该操纵子5'端的核苷酸序列，包括控制区、glpF基因和部分glpK基因。预测该转运蛋白含有281个氨基酸，计算分子量为29,780。它是一种高度疏水的蛋白质，至少有六个潜在的跨膜α螺旋。glpFK操纵子的转录起始位点位于glpF推测的翻译起始密码子上游71个碱基对处。在转录起始位点之前的序列与细菌启动子的-10和-35共有序列相似。通过DNA酶I足迹法鉴定了cAMP - cAMP受体蛋白（CRP）复合物和glp阻遏物的结合位点。cAMP·CRP复合物保护的区域包含类似于CRP结合共有序列的串联序列。CRP位点位于转录起始点上游37.5和60.5个碱基对处。glp阻遏物保护了一个广泛的区域（相对于转录起始点为-89至-7），足以结合四个阻遏物四聚体。在glpK编码区内还鉴定出另外两个阻遏物结合位点。含有这两个操纵基因的DNA协同增加了glp阻遏物对glpFK操纵子启动子区域含有四个操纵基因的DNA片段的表观亲和力。通过这项研究，总共鉴定了13个glp调节子的操纵基因。对这些操纵基因的比较揭示了操纵基因半位点的共有序列5'-WATGTTCGWT-3'（W = A或T）。使用启动子探针载体在体内评估了glp阻遏物对各种glp操纵基因的相对亲和力。控制区对glp阻遏物的相对表观亲和力为glpFK大于glpD大于glpACB大于glpTQ。使用类似系统评估了每个操纵子的分解代谢物阻遏程度。在这种情况下，glp操纵子对分解代谢物阻遏的相对敏感性为glpTQ大于glpFK大于glpACB大于glpD。
