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Nucleic Acids Research, 1987, Vol. 15, No. 21 8773-8781
© 1987

Articles

Specific labelling of the active site of T7 RNA polymerase

Anton R. Schäffner, Ellen D. Jorgensen1, William T. McAllister1 and Guido R. Hartmann

Institut Für Biochemie, Ludwig-Maximilians-Universität D-8000 München 2, FRG 1SUNY-Health Science Center, Morse Institute for Human and Molecular Genetics, Department of Microbiology and Immunology 450 Clarkson Avenue, Brooklyn, NY 11203, USA

Received July 17, 1987. Revised October 1, 1987. Accepted October 1, 1987.

We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.


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