Transcription initiation at the tryptophanase promoter of Escherichia coli K-12

Abstract

Restriction fragments containing the region preceding the tryptophanase structural gene, tnaA, were used as templates for in vitro transcription experiments. A transcription initiation site was detected that was dependent on the catabolite gene activator protein (CAP) plus cyclic AMP (cAMP). The mRNA produced in vitro was fingerprinted, and the nucleotide at which transcription was initiated was localized to the vicinity of two guanine residues 316 and 318 base pairs upstream of tnaA. A region exhibiting extensive difold symmetry and homology to the CAP binding site adjacent to the lactose operon promoter exists approximately 60 base pairs preceding the site of transcription initiation. Two HinfI restriction sites are located in this region. Restriction enzyme cleavage at these sites was prevented when DNA containing the promoter region was preincubated with CAP and cAMP. RNA polymerase was incapable of protecting these sites against this cleavage. CAP and cAMP addition did not protect against cleavage at a DdeI restriction site located in the -20 region of the promoter. RNA polymerase did protect against DdeI cleavage but only in the presence of CAP and cAMP. Thus, transcription initiation at the tryptophanase promoter involves cAMP-dependent, CAP-facilitated binding of RNA polymerase to the DNA.