EFFECTOR-MEDIATED STIMULATION OF ATPASE ACTIVITY BY THE SIGMA(54)-DEPENDENT TRANSCRIPTIONAL ACTIVATOR FHLA FROM ESCHERICHIA-COLI

The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli, The protein shares homology wit h the sigma(54)-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N termin us lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA, The presence of both formate and DNA carryi ng the upstream regulatory sequence to which FHLA binds leads to a lar ge increase in the rate of ATP hydrolysis, Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, a lso stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA, Half-maximal saturation of FHLA,vith forma te took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM, The stimulation of ATPase activity by formate was conferred by a decrease in the apparent K-m for ATP, where as the effect of the DNA binding site also affected the K-cat of the r eaction, The other nucleoside triphosphates, GTP, UTP, and CTP, compet ed with ATP cleavage by FHLA, suggesting at least their binding to FHL A. The specific ATPase activity of FHLA was dependent on the concentra tion of FHLA in the assay, especially in the presence of DNA and forma te, Direct liganding of the effector, therefore, leads to the same con sequence as phosphorylation for the NTRC-type regulators, namely, stim ulation of ATPase activity.