A genetic method for dissecting the mechanism of transcriptional activatorsynergy by identical activators

Pairs of transcriptional activators in prokaryotes have been shown to activ ate transcription synergistically from promoters with two activator binding sites. In some Eases, such synergistic effects result from cooperative bin ding, but in other cases each DNA-bound activator plays a direct role in th e activation process by interacting simultaneously with separate surfaces o f RNA polymerase. In such Eases, each DNA-bound activator must possess a fu nctional activating region, the surface that mediates the interaction with RNA polymerase, When transcriptional activation depends on two or more iden tical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method fo r directing a mutationally altered activator to either one or the other bin ding site, and we demonstrate the use of this method to examine the mechani sm of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-bi nding sites.