Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences

The Escherichia coli Trp repressor binds to promoters of very different seq uence and intrinsic activity. Its mode of binding to trp operator DNA. has been studied extensively yet remains highly controversial. In order to exam ine the selectivity of the protein for DNA, we have used electromobility sh ift assays (EMSAs) to study its binding to synthetic DNA containing the cor e sequences of each of its five operators and of operator variants. Our res ults for DNA containing sequences of two of the operators, trpEDCBA and aro H are similar to those of previous studies. Up to three bands of lower mobi lity than the free DNA are obtained which are assigned to complexes of stoi chiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and ar oL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mo bility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previousl y obtained with super-repressor Trp mutants. This gives bands with mobiliti es corresponding to 1:1 and 2:1 complexes. In contrast, DNA containing cont aining a symmetrized trpR operator sequence, trpR(5), gives a single retard ed band with mobility corresponding solely to a 1 : 1 protein dimer-DNA com plex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands i n EMSA and the length of the DNase I footprint observed. This shows that th e binding of the second dimer is sequence selective. We propose that the br oad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor t o bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This me chanism may be of importance in a number of promoters that bind multiple ef fector molecules.