title: 8-Azido-ATP inactivation of Escherichia coli transcription termination factor Rho. Modification of one subunit inactivates the hexamer.
abstract: Escherichia coli transcription termination factor Rho (EC 3.6.1.3) releases nascent RNA from transcription complexes in a reaction which requires ATP hydrolysis. To understand the structure of the ATPase active site, we employed an analog of ATP, 8-azidoadenosine 5'-triphosphate (8-azido-ATP) as a photoaffinity labeling agent. 8-Azido-ATP interacts nearly normally with the active site of Rho. It binds to 3 sites per Rho hexamer with a 100 microM KD and is a substrate with a Vmax 5% that of ATP and a Km of 18 microM. Under UV irradiation, 8-azido-ATP makes covalent bonds with Rho, inactivating its ATPase. Rho is protected from this inactivation by the presence of ATP. We used [alpha-32P]8-azido-ATP to label the active site and identify residues involved in ATP binding. Labeled tryptic peptides of the modified Rho were purified by Fe(3+)-iminodiacetic acid affinity chromatography and reverse-phase C18 column high performance liquid chromatography. We identified a single peptide, Gly174-Lys184, that is labeled by 8-azido-ATP and protected from labeling in the presence of ATP. The modified amino acid is Lys181, whose conservative replacement by Gln181 gives rise to a poorly active enzyme (Dombroski, A. J., Brennan, C. A., Spear, P., and Platt, T. (1988a) J. Biol. Chem. 263, 18802-18809). Lys181 probably participates in binding the phosphoryl groups of ATP. Incorporation of one 8-azido-ATP per Rho hexamer is sufficient to cause inactivation, a result that indicates that the active sites of Rho interact in RNA-dependent ATP hydrolysis.
authors.affiliation: Department of Biology, New York University, Washington Square, New York 10003.
authors.forename: I
authors.initials: I
authors.lastname: O
authors.forename: B L
authors.initials: BL
authors.lastname: Stitt
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010782:Photolysis; D011485:Protein Binding; D012234:Rho Factor / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013997:Time Factors; D014357:Trypsin
chemical_list: D001386:Azides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012234:Rho Factor; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin