The Escherichia coli MelR transcription activator: production of a stable fragment containing the DNA-binding domain.
A set of nested deletions has been made in the Escherichia coli melR gene, encoding the MelR transcription activator protein. Expression of the resulting melR derivatives led to the production of nine MelR proteins with N-terminal deletions of different lengths. The properties of the shortened proteins have been studied both in vivo and in vitro. None of the truncated proteins activate transcription from the E.coli melAB promoter but three; MelR220, MelR183 and MelR173, inhibit activation of the melAB promoter by chromosomally-encoded full-length MelR. In gel retardation assays, both MelR183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MelR173 has been overproduced in a T7 expression system and shown to be stable in vivo for up to 2 h. DNAase I footprinting assays of partially purified protein show that it binds to the melAB promoter, protecting the same sites as the full-length protein. This fragment may be suitable for further structure/function studies of this class of transcription activator.
Michán
C M
CM
School of Biochemistry, University of Birmingham, UK.
Busby
S J
SJ
Hyde
E I
EI
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C077649:MelR protein, E coli; D011994:Recombinant Proteins; D015534:Trans-Activators