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 Escherich ia coli. The cloned FhlA gene was overexpressed under selected growth conditions, and a purification protocol for the FHLA protein was devel oped. Purified FHLA in the native state is a homotetramer. It binds to the upstream regulatory sequences of the fdhF gene and of the interge nic region between the divergently transcribed hyc and hyp operons. An additional binding site of FHLA located between the hycA and hycB gen es 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)-depen dent promoter located upstream of the hyp operon. Formate seems to hav e no effect on the DNA binding properties of FHLA. The binding sites o f FHLA were characterized by DNase I footprinting; sequence motifs put atively involved in the interaction with FHLA are described.