Expression and characterisation of the korB gene product from the Streptomyces lividans plasmid pIJ101 in Escherichia coli and determination of its binding site on the korB and kilB promoters.
A 0.5kb Spel-BclI fragment containing the pIJ101 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. coli. Crude E. coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays. DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter.
Zaman
S
S
Department of Biology, University College, London, UK.
Richards
H
H
Ward
J
J
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C053863:KorB protein, Plasmid RK2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C069003:kil protein, E coli