Two amino acid residues from the DNA-binding domain of MalT play a crucial role in transcriptional activation.
MalT is the transcriptional activator of the Escherichia coli maltose regulon. Several lines of evidence suggest that MalT might act by interacting with RNA polymerase. Here, we show that 'MalT, the DNA-binding domain of MalT, activates transcription. In order to identify amino acids of 'MalT playing a specific role in activation, and therefore possibly involved in the putative contact(s) with RNA polymerase, we developed a double screen to isolate mutations of the 'malT gene affecting activation by 'MalT without impairing its DNA-binding affinity. The effect of the mutations thus obtained on activation was assessed in vivo. This strategy essentially pointed to serine 834 and glutamine 876 of the MalT amino acid sequence as specifically involved in activation. Various 'MalT derivatives substituted at positions 834 or 876 were purified and tested in vitro for their DNA-binding affinity, as well as for their activation ability. Together, the results obtained clearly show that serine 834 and glutamine 876 are important for activation by 'MalT but not for DNA-binding. We argue that these amino acid residues are possibly solvent-exposed and propose that they act by contacting RNA polymerase.
Danot
O
O
Unité de Génétique Moléculaire, (CNRS URA 1149), Institut Pasteur, Paris, France.
Vidal-Ingigliardi
D
D
Raibaud
O
O
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058976:MalT protein, E coli; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase