Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal -35 region sequences.
The gal operon regulatory region contains two overlapping promoters, P1 and P2, regulated by cyclic AMP and the cyclic AMP receptor protein (cAMP X CRP). Starting with a mutation that eliminated P1, the promoter that is usually dependent on cAMP X CRP, we constructed a series of deletions that substituted increasing amounts of DNA sequence from upstream of P2, the promoter that usually functions in the absence of cAMP X CRP. Expression from P2 in vivo was halved by deletions that replace the -35 region with unrelated sequences, showing that the -35 sequence participates in promoter function, but is not essential. In vitro studies show that replacement of the -35 sequence increases the time for open complex formation at P2, but does not alter the transcription start point. We examined the effects of the same deletions at the wild type gal promoter region: again, the deletion that replaces the -35 region halves expression in vivo. However, in this case, in the absence of cAMP X CRP, the deletion switches expression from the P2 promoter to P1, the promoter that is usually dependent on cAMP X CRP. Moreover, although the deletion also removes the specific cAMP X CRP binding site, this P1 activity is sharply inhibited in a crp+ background. We argue that this is due to a direct contact between CRP and RNA polymerase bound at the P1 Pribnow box, and we discuss the role of the -35 sequence at these and other promoters.
D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics*; D005690:Galactose / Q000235:genetics*; D007700:Kinetics; D009876:Operon; D011401:Promoter Regions, Genetic; D011953:Receptors, Cyclic AMP / Q000502:physiology; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D011953:Receptors, Cyclic AMP; D001616:beta-Galactosidase; D005690:Galactose