Dual mechanism of repression at a distance in the lac operon.

The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants. Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site. The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression. This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold. Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements. During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously.