Mechanism of a trans Dominant Negative Mutation

Mechanism of a trans Dominant Negative Mutation

Expression of the lac operon is regulated by the product of the lacI gene. Since this protein acts to turn off or turn down the expression, it is called a repressor. The protein can bind to a site partially overlapping the lac promoter and block transcription from the promoter into the lac struc-tural genes. In the presence of inducers, the repressor’s affinity for the operator is much reduced and it dissociates from the DNA. This then permits active transcription of the lac genes.

The repressor contains four identical subunits. Consider cells that are diploid for lacI where one of the lacI genes is lacI⁺, and the other is a type we call lacI^-d. The d stands for “dominant.” During the synthesis of the two types of repressor subunit in a cell, the probability that four newly synthesized wild-type repressor subunits will associate to form a wild-type tetramer is low. Instead, most repressor tetramers will possess both types of subunits.

If the inclusion of a single I^-d subunit in a tetramer interferes with the function of a tetramer, then the I^-d allele will be dominant and act in trans to nullify the activity of the good lacI allele, that is, it is a trans dominant negative mutation.

What is the physical basis for a single defective subunit inactivating the remaining three nondefective subunits in a tetramer? Lac repressor contacts the symmetrical lac operator with two subunits utilizing the helix-turn-helix structure that was discussed. Contact with only one subunit provides far too little binding energy for the protein to bind. Therefore a subunit with a defective DNA-contacting domain may be capable of folding and oligomerizing with normal subunits, but its incorporation into a tetramer will interfere with DNA binding since two good subunits must simultaneously be involved in contacting the opera-tor.

If a tetramer contains only one defective subunit, we might think that two nondefective subunits could still be utilized for binding to DNA. To an extent this is true. However, just as enhancers loop and complexes of proteins can contact the DNA at two or more sites, so does the lac

Figure 8.6 When all four subunits oflacrepressor can bind DNA and form theloop, binding to both of the DNA sites is enhanced because looping effectively increases the local concentration of the repressor at the other sites.

operon. Looping in the lac operon brings repressor bound at the primary operator into contact with either of two so-called pseudo-operators on either side. As will be discussed later, such looping can greatly increase the occupancy of a binding site by a protein. In the case of the tetrameric lac repressor, two good subunits could contact the lac operator or apseudo operator, but looping could not occur, and the overall binding would be relatively weak and as a result, repression would be poor (Fig. 8.6). Thus, the inclusion of a single DNA-binding defective subunit in a tetrameric repressor can greatly interfere with repression.