Fritz H J, Bicknäse H, Gleumes B, Heibach C, Rosahl S, Ehring R
EMBO J. 1983;2(12):2129-35. doi: 10.1002/j.1460-2075.1983.tb01713.x.
A series of plasmids has been constructed which contain either a single one of the two operators O1 and O2 of the Escherichia coli galactose operon or different combinations thereof. This permits comparison of the two operators with respect to their repressor binding ability. A plasmid containing only the second operator, O2, located within the structural gene galE, was found to titrate repressor in vivo and in vitro with essentially the same efficiency as a plasmid containing only the 'classical' galactose operator, O1, located upstream of the start of transcription. Whereas some cooperativity between the two sites seems possible in vivo, they bind repressor independently under the conditions of the in vitro assay. After hydroxylamine treatment of plasmid DNA in vitro, two different mutations have been isolated each of which inactivates the second operator, O2. Both are GC to AT transitions located at equivalent positions relative to the axis of rotational symmetry within the second gal operator. Subdividing O1 and O2 according to their 2-fold symmetry yields four half-sites with the consensus sequence (5')gTGnaAnC(3'). All known single point mutations of O1 and O2 affect the frame of invariant residues. The two half-sites of the lac operator also coincide with this frame.
构建了一系列质粒，这些质粒要么包含大肠杆菌半乳糖操纵子的两个操纵基因O1和O2中的单个操纵基因，要么包含它们的不同组合。这使得能够比较这两个操纵基因的阻遏物结合能力。发现一个仅包含位于结构基因galE内的第二个操纵基因O2的质粒，在体内和体外滴定阻遏物的效率与一个仅包含位于转录起始上游的“经典”半乳糖操纵基因O1的质粒基本相同。虽然在体内两个位点之间似乎可能存在某种协同作用，但在体外测定条件下它们独立结合阻遏物。在体外对质粒DNA进行羟胺处理后，分离出了两种不同的突变，每种突变都使第二个操纵基因O2失活。两者都是GC到AT的转换，位于第二个半乳糖操纵基因内相对于旋转对称轴的等效位置。根据O1和O2的二重对称性进行细分，可得到四个具有共有序列(5')gTGnaAnC(3')的半位点。所有已知的O1和O2的单点突变都会影响不变残基的框架。乳糖操纵基因的两个半位点也与这个框架一致。
