Specific labelling of the active site of T7 RNA polymerase.
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.
Institut für Biochemie, Ludwig-Maximilians-Universität, München, FRG.
A R
AR
Schäffner
E D
ED
Jorgensen
W T
WT
McAllister
G R
GR
Hartmann
D000345:Affinity Labels; D001665:Binding Sites; D004259:DNA-Directed DNA Polymerase; D006150:Guanine Nucleotides; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes; D013604:T-Phages / Q000201:enzymology*
D000345:Affinity Labels; D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase