A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase.
We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.
D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010455:Peptides / Q000032:analysis*
D046911:Macromolecular Substances; D010455:Peptides; D012321:DNA-Directed RNA Polymerases