Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA.
FhlA is the transcriptional activator of the genes coding for the formate hydrogen lyase system in Escherichia coli. It is activated by the binding of formate and induces transcription by sigma54 RNA polymerase after binding to specific upstream activating sequences (UAS). Sequence comparison had shown that FhlA exhibits a structure composed of three domains, which is typical for sigma54-dependent regulators. By analyzing the N-terminal domain of FhlA of E. coli (amino acids 1-378; FhlA-N) and the rest of the protein (amino acids 379-693; FhlA-C) as separate proteins in vivo and in vitro the functions of the different domains of FhlA were elucidated. The FhlA-C domain is active in ATP hydrolysis and activation of transcription and its activity is neither influenced by the presence of formate nor of the antiactivator HycA. However, it is stimulated in the presence of the FhlA-specific UAS, indicating that this region of FhlA is responsible for DNA binding. FhlA-N is not active itself but able to reduce the activity of full-length FhlA in trans, probably by formation of nonfunctional heterooligomers. The DNA binding site of FhlA was analyzed by hydroxyradical footprinting. Each UAS consists of two binding sites of 16 bp separated by a spacer region. A consensus sequence could be deduced and a model is presented and supported by in vivo data in which a FhlA tetramer binds to the UAS on one side of the DNA helix. Performing an extensive screening we could show that the FhlA regulatory system is conserved in different species of the family Enterobacteriaceae. The analysis of orthologs of FhlA revealed that they are able to functionally replace the E. coli enzyme.
Leonhartsberger
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Lehrstuhl für Mikrobiologie der Universität München, Germany.
Ehrenreich
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Böck
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004754:Enterobacter / Q000235:genetics / Q000378:metabolism; D004755:Enterobacteriaceae / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D017665:Hydroxyl Radical / Q000378:metabolism; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D015534:Trans-Activators; C067189:fhlA protein, E coli; D017665:Hydroxyl Radical; D004247:DNA