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Nucleic Acids Research, 1992, Vol. 20, No. 14 3693-3700
© 1992

MOLECULAR BIOLOGY

Expression and characterisation of the korB gene product from the Streptomyces lividans plasmid plJ101 in Escherichia coli and determination of its binding site on the korB and kilB promoters

Sara Zaman, Hilary Richards and John Ward1,*

Department of Biology Gower Street, London WC1E 6BT, UK 1Department of Biochemistry and Molecular Biology, University College London Gower Street, London WC1E 6BT, UK

*To whom correspondence should be addressed

Received March 20, 1992. Revised June 24, 1992. Accepted June 24, 1992.

A 0.5kb Spel-Bc/1 fragment containing the plJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206. In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence. pQR206 was used to express the 10kDa korB protein in vivo in E. coif. Crude E. coll protein extracts containing korB were shown to bind to a 0.8kb kllB fragment and a 0.5kb korB fragment in gel retardation assays. DNasel footprintlng Indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kllB promoter and a 36bp region encompassing the korB promoter.


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