Biochemical Investigations Verify the Operon Hypothesis

H Genetic studies led to the operon hypothesis. Biochemical investigations were essential to provide direct evidence for the hypothesized properties of the repressor. The first task was to isolate the repressor. 

Several properties of normal and abnormal repressor were studied. The repressor was found to bind strongly to the lac promoter. This binding was disrupted by adding inducer. Promoter DNA containing an oc mutation was not effective in binding repressor. Only repressor prepared from i+ cells was effective in binding to DNA. 

The sequence of bases in the operator region shows a remarkable symmetry property. Twenty-eight out of 36 of the base pairs in this region of the promoter are located on a twofold (dyad) axis of symmetry (see fig. 30.7). We shall see that dyad symmetry is a common property for repressor or activator binding sites and that it relates to the way in which DNA and regulatory proteins interact. 

Biochemical proof that repressor inhibits operon expression was shown in a cell-free system in which most of the components were prepared from an i extract. Addition of repressor to such an extract inhibited the synthesis of j8-galactosidase. This inhibition was reversed by addition of IPTG inducer. 

The detailed biochemical studies not only confirmed the Jacob-Monod hypothesis, they gave further details about the nature of repressor interaction and a detailed characterization of the repressor binding site. The repressor binds mainly to one side of the double helix, over a 36-base region covered by the symmetry axis. It inhibits expression because it binds to a site that overlaps the polymerase binding site. 

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