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Nucleic Acids Research, 1995, Vol. 23, No. 9 1518-1523
© 1995

MOLECULAR BIOLOGY

The Escherichia coli MeIR transcription activator: production of a stable fragment containing the DNA-binding domain

Carmen M. Michan+, Stephen J. W. Busby and Eva I. Hyde*

School of Biochemistry, University of Birmingham PO Box 363, Birmingham B15 2TT, UK

*To whom correspondence should be addressed

Received January 23, 1995. Revised March 17, 1995. Accepted March 17, 1995.

A set of nested deletions has been made In the Escherichia coll melR gene, encoding the MelR transcription activator protein. Expression of the resulting meIR derivatives led to the production of nine MeIR proteins with N-termlnal 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.coll melAB promoter but three; MelR220, MelR183 and MelR173, Inhibit activation of the melAB promoter by chromoso-mally-encoded full-length MeIR. In gel retardation assays, both MeIR 183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MeIR 173 has been overproduced In a T7 expression system and shown to be stable In vivo for up to 2 h. DNAase I footprintlng 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.

+Present address: CSIC Estacion Experimental del Zaidin, C. Prof. Albareda 1, 18080, Granada, Spain


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