title: Effector-mediated stimulation of ATPase activity by the sigma 54-dependent transcriptional activator FHLA from Escherichia coli.
abstract: The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus 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 carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate 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 Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität München, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Hopper
authors.forename: A
authors.initials: A
authors.lastname: Böck
mesh_terms: D000251:Adenosine Triphosphatases / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005561:Formates / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010721:Phosphinic Acids / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005561:Formates; D009838:Oligodeoxyribonucleotides; D010721:Phosphinic Acids; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; C030544:formic acid; C067189:fhlA protein, E coli; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000251:Adenosine Triphosphatases