FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter.
The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.
Muskhelishvili
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G
Institute für Genetik und Mikrobiologie, München, Germany.
Travers
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AA
Heumann
H
H
Kahmann
R
R
D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009698:Nucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000378:metabolism*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D009698:Nucleoproteins; D012329:RNA, Bacterial; C440502:integration host factor, E coli; D012321:DNA-Directed RNA Polymerases