Mutagenesis of the downstream region of the Escherichia coli hns promoter.
The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene. To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence. The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small. Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels.
Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy.
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Giangrossi
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Gualerzi
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Pon
D001426:Bacterial Proteins / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003080:Cold Temperature; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D016296:Mutagenesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase