Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli.
The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS. Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription. We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting. The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro. We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex. Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs. The implications of our results for the osmotic regulation of proU transcription are discussed.
Lucht
J M
JM
Department of Biology, University of Konstanz, Germany.
Dersch
P
P
Kempf
B
B
Bremer
E
E
D026905:Amino Acid Transport Systems; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping
D026905:Amino Acid Transport Systems; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; C060774:ProU protein, Bacteria