Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit.
Activation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family. MelR binds to 18 bp target sites using two helix-turn-helix (HTH) motifs that are both located in its C-terminal domain. The melAB promoter contains four target sites for MelR. Previously, we showed that occupation of two of these sites, centred at positions -42.5 and -62.5 upstream of the melAB transcription start point, is sufficient for activation. We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned. Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site. As our model for DNA-bound MelR suggests that HTH 2 must be adjacent to the melAB promoter -35 element, we searched this part of MelR for amino acid side-chains that might be able to interact with sigma. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase sigma(70) subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of sigma(70). In the final part of the work, we describe experiments in which the MelR binding site at position -42.5 was improved. We show that this is detrimental to MelR-dependent transcription activation because bound MelR is mispositioned so that it is unable to make 'correct' interactions with sigma.
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
David C
DC
Grainger
Christine L
CL
Webster
Tamara A
TA
Belyaeva
Eva I
EI
Hyde
Stephen J W
SJ
Busby
D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009876:Operon; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C077649:MelR protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; D012321:DNA-Directed RNA Polymerases; D000409:Alanine