Mapping of the active site of T7 RNA polymerase with 8-azidoATP

The photoaffinity analog of ATP, 8-azidoATP, labels T7 RNA polymerase. Photoincorporation exhibits saturation behavior and is protected against by the substrate ATP. 8-AzidoATP is a competitive inhibitor of ATP incorporation with Ki ∼ 40 μM. The photolabeled T7 RNA polymerase, following cyanogen bromide digestion, was analyzed by phenylboronate agarose column chromatography followed by reverse-phase high pressure liquid chromatography. Sequencing of the peptides labeled with radioactive photoprobe allowed the identification of three peptides, P314-M362 (I), L550-M666 (II), and F751-M861 (III). These peptides are in the proximity of the photoprobe 8-azidoATP and, therefore, expected to contain functionally significant residues and define an active site domain. These peptides (I and II) contain residues previously implicated in T7 RNA polymerase activity or show homology to active site regions of the Klenow fragment of DNA polymerase I (II and III).