A sensitive single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis

摘要

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the Km value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was >∼100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3′-deoxy-ATP. Whereas the Ki value for 3′-deoxy-ATP (105–117 >µM) is similar to the Km value for ATP (186 µM) in the case of influenza RNA polymerase, the Ki value for 3′-deoxy-ATP (0.4>–0.6 >µM) is ∼100-fold lower than the Km value for ATP (50 >µM) in the case of yeast PAP.