Polyadenylation of RNA in E. Coli: RNA polymerase-associated (rA)n-Synthetic activities

Abstract

In Escherichia coli, Poly(A) polymerase (PAP) and polynucleotide phosphorylase (PNP) are key enzymes thought to be responsible for polyadenylation of the bulk of cellular RNA. In this chapter we describe enzymatic in vitro assays for monitoring (rA) n -synthetic activity among fractionated E. coli proteins obtained after affi nity chromatography on immobilized DNA. The enzymatic activities of PAP and PNP can be independently monitored among fractionated proteins due to the utilization of different nucleoside substrates (respectively, ATP and ADP) by the two enzymes. We describe two different methods for monitoring the synthesis of polyadenylate: a method based on utilization of a nucleic acid-specifi c fl uorescent dye (RiboGreen ®) and an alternative method based on utilization of P 32 -labeled nucleoside phosphates.