8-Azido-ATP inactivation of Escherichia coli transcription termination factor Rho. Modification of one subunit inactivates the hexamer.
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.
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Department of Biology, New York University, Washington Square, New York 10003.
Stitt
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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
D001386:Azides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012234:Rho Factor; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin