Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli.
FHLA is the transcriptional activator for the expression of the genes coding for components of the formate hydrogenlyase system of Escherichia coli. The cloned fhlA gene was overexpressed under selected growth conditions, and a purification protocol for the FHLA protein was developed. Purified FHLA in the native state is a homotetramer. It binds to the upstream regulatory sequences of the fdhF gene and of the intergenic region between the divergently transcribed hyc and hyp operons. An additional binding site of FHLA located between the hycA and hycB genes was identified. While binding to the hypA-hycA intergenic region is responsible for activation of expression of the hyc operon, the newly identified site is required for the activation of the sigma 54-dependent promoter located upstream of the hyp operon. Formate seems to have no effect on the DNA-binding properties of FHLA. The binding sites of FHLA were characterized by DNase I footprinting; sequence motifs putatively involved in the interaction with FHLA are described.
Schlensog
V
V
Lehrstuhl für Mikrobiologie der Universität München, Germany.
Lutz
S
S
Böck
A
A
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005560:Formate Dehydrogenases / Q000235:genetics*; D006864:Hydrogenase / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics; D009876:Operon / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C067189:fhlA protein, E coli; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase