DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation.
The betT and betIBA genes govern glycine betaine synthesis from choline in Escherichia coli. In an accompanying paper we report that the betT and betI promoters are divergently organized and partially overlapping and that both are negatively regulated by BetI in response to choline. (T. Lamark, T.P. Rokenes, J. McDougall, and A.R. Strom, J. Bacteriol. 178:1655-1662, 1996). In this paper, we report that the in vivo synthesis rate of the BetI protein constituted only 10% of that of BetA and BetB dehydrogenase proteins, indicating the existence of a posttranscriptional control of the betIBA operon. A genetically modified BetI protein called BetI*, which carries 7 extra N-terminal amino acids, was purified as a glutathione S-transferase fusion protein. Gel mobility shift assays showed that BetI* formed a complex with a 41-bp DNA fragment containing the -10 and -35 regions of both promoters. Only one stable complex was detected with the 41-bp fragment and all larger promoter-containing fragments tested. In DNase I footprinting, BetI* protected a region of 21 nucleotides covering both the -35 boxes. Choline stimulated complex formation but did not change the binding site of BetI*. We conclude that in vivo BetI is bound to its operator in both repressed and induced cells and that BetI represents a new type of repressor.
Rkenes
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Norwegian College of Fishery Science, University of Tromsø, Norway.
Lamark
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Strøm
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D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002794:Choline / Q000494:pharmacology; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; C098629:BetI protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D002794:Choline