Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of the E. coli galactose operon

Abstract

We have examined the interaction site on gal DNA for the cyclic AMP receptor protein and RNA polymerase when both are present together to form a stable initiation complex at the P1 gal promoter. Substitution of the bases to the left of -60 by unrelated DNA sequences does not change the cyclic AMP concentration dependency for in vitro transcription at P1 and inhibition of P2. Although the presence of some DNA to the left of -60 appears to be needed for efficient in vitro transcription at P1, the gal sequence to the left of -60 does not provide any specific interactions for transcription initiation at P1. Similarly, efficient in vitro transcription from P2 also requires non-specific DNA sequences to the left of -60. We have also examined which bases were protected by RNA polymerase and CRP together from the action of DNAase and dimethylsulfate. Some of the interactions that take place when cAMP-CRP alone interacts with gal DNA appear to be preserved in the cAMP-CRP-RNA polymerase-gal DNA complex, suggesting that CRP occupies the same site in the DNA when it is alone or together with RNA polymerase. Our results suggest that the formation of an open complex at different promoters can result from different interaction patterns between RNA polymerase and promoter DNA.