1610 The partial diploid lacIs Iac ZlacI lacZ fails to produce pgalactosidase in the presence and absence of lactose because the lacIs gene encodes a superrepressor

The lacIs gene is trans dominant: the superrepressor binds both operators and prevents transcription in the presence and absence of lactose.

Operator mutations Jacob and Monod mapped the other class of constitutive mutants to a site adjacent to lacZ. These mutations occurred at the operator site, and were labeled lacOc (O stands for operator and c for constitutive). The lacOc mutations altered the sequence of DNA at the operator so that the repressor protein was no longer able to bind. A partial diploid with genotype lacl+ lacOc lacZ+/lacI+ lacO+ lacZ+ exhibited constitutive synthesis of p-galactosidase, indicating that lacOc was dominant over lacO+.

Analysis of other partial diploids showed that the lacO gene was cis acting, affecting only genes on the same DNA molecule. For example, a partial diploid with genotype lacI+ lacO+ lacZ"/lacI+ lacOc lacZ+ was constitutive, producing p-galactosidase in the presence or absence of lactose (Figure 16.11a), but a partial diploid with genotype lacI+ lacO+ lacZ+/lacI+ lacOc lacZ" produced p-galactosidase only in the presence of lactose (Figure 16.11b). In the constitutive partial diploid (lacI+ lacO+ lacZ"/lacI+ lacOc lacZ+; see Figure 16.11a), the lacOc mutation and the functional lacZ+ gene are present on the same DNA molecule; but in lacI+ lacO+ lacZ+/lacI+ lacOc lacZ" (see Figure 16.11b), the lacOc mutation and the functional lacZ+ gene are on different molecules. The lacO mutation affects only genes to which it is physically connected, as is true of all operator mutations. They prevent the binding of a repressor protein to the operator and thereby allow RNA polymerase to transcribe genes on the same DNA molecule. However, they cannot prevent a repressor from binding to normal operators on other DNA molecules.

Promoter mutations Mutations affecting lactose metabolism have also been isolated at the promoter site; these mutations are designated lacP", and they interfere with the binding of RNA polymerase to the promoter. Because this binding is essential for the transcription of the structural genes, E. coli strains with lacP" mutations don't produce lac enzymes either in the presence or in the absence of lactose. Like operator mutations, lacP" mutations are cis acting and affect only genes on the same DNA molecule. The partial diploid lacI+ lacP+ lacZ+/lacI+ lacP" lacZ+ exhibits normal synthesis of p-galactosidase, whereas the lacI+ lacP" lacZ+/lacI+ lacP+ lacZ" fails to produce p-galactosidase whether or not lactose is present.

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