Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein

MelR is a melibiose-triggered transcription activator that belongs to the A raC family of transcription factors. Using purified Escherichia coli RNA po lymerase and a cloned DNA fragment carrying the entire melibiose operon int ergenic region, we have demonstrated in vitro open complex formation and ac tivation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cycli c AMP receptor protein (CRP) interacts with the melAB promoter and increase s MelR-dependent transcription activation. DNAase I footprinting has been e xploited to investigate the location of MelR-and CRP-binding sites at the m elAB promoter. We showed previously that MelR binds to two identical 18 bp target sequences centred at position -100.5 (Site 1) and position -62.5 (Si te 2). In this work, we show that MelR additionally binds to two other rela ted 18 bp sequences: Site 1', centred at position -120.5, located immediate ly upstream of Site 1, and Site R, at position -238.5, which overlaps the t ranscription start site of the divergent melR promoter. MelR can bind to Si te 1', Site 1, Site 2 and Site R, in both the absence and the presence of m elibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream o f Site 2, and overlapping the -35 region of the melAB promoter. Additionall y, although CRP is unable to bind to the melAB promoter in the absence of M elR, in the presence of MelR, it binds to a site located between MelR bindi ng Site 1 and Site 2. Thus, tandem-bound MelR recruits CRP to the MelR. We propose that expression from the melAB promoter has an absolute requirement for MelR binding to Site 2'. Optimal expression of the melAB promoter requ ires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between M elR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression fr om the melAB promoter. In support of this model, we show that improvement o f the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP.