Characterization of two mutations in the Escherichia coli galE gene inactivating the second galactose operator and...

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.