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 AraC family of transcription factors. Using purified Escherichia coli RNA polymerase and a cloned DNA fragment carrying the entire melibiose operon intergenic region, we have demonstrated in vitro open complex formation and activation of transcription initiation at the melAB promoter. This activation is dependent on MelR and melibiose. These studies also show that the cyclic AMP receptor protein (CRP) interacts with the melAB promoter and increases MelR-dependent transcription activation. DNAase I footprinting has been exploited to investigate the location of MelR-and CRP-binding sites at the melAB 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 (Site 2). In this work, we show that MelR additionally binds to two other related 18 bp sequences: Site 1', centred at position -120.5, located immediately upstream of Site 1, and Site R, at position -238.5, which overlaps the transcription start site of the divergent melR promoter. MelR can bind to Site 1', Site 1, Site 2 and Site R, in both the absence and the presence of melibiose. However, in the presence of melibiose, MelR also binds to a fifth site (Site 2', centred at position -42.5) located immediately downstream of Site 2, and overlapping the -35 region of the melAB promoter. Additionally, although CRP is unable to bind to the melAB promoter in the absence of MelR, in the presence of MelR, it binds to a site located between MelR binding 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 requires Sites 1', Site 1, Site 2 and Site 2'; CRP acts as a 'bridge' between MelR bound at Sites 1' and 1 and at Sites 2 and 2', increasing expression from the melAB promoter. In support of this model, we show that improvement of the base sequence of Site 2' removes the requirement for Site 1' and Site 1, and short circuits the effects of CRP.
Belyaeva
T A
TA
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Wade
J T
JT
Webster
C L
CL
Howard
V J
VJ
Thomas
M S
MS
Hyde
E I
EI
Busby
S J
SJ
D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D027981:Symporters; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation
D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C077649:MelR protein, E coli; D026901:Membrane Transport Proteins; D027981:Symporters; D015534:Trans-Activators; C021548:melibiose permease; D008553:Melibiose; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I