A simple method for measuring specific radioactivities of ribonucleoside triphosphates using RNA polymerase.

ABSTRACT

We describe a method for the rapid, one-step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP. Escherichia coli RNA polymerase and poly[d(A-T)] or poly[d(G-C)] are used to synthesize an alternating copolymer from a [3H]nucleoside triphosphate of unknown specific activity and a [14C]nucleoside triphosphate of known specific activity. The fact that [3H]nucleotide and [14C]nucleotide are incorporated into poly[r(A-U)] or poly[r(G-C)] in equimolar amounts, coupled with a knowledge of the [14C]nucleotide specific activity, permits calculation of the [3H]nucleotide specific activity. The requirement for direct knowledge of the [14C]nucleotide specific activity may be bypassed by an isotope dilution procedure. The pool size of a nucleoside triphosphate can be estimated either from isotope dilution data or by determining the fraction of [3H]nucleotide polymerized, dividing the number of counts 3H/min in the polymer by this fraction and by the [3H]nucleotide specific activity. The method was successfully applied to acid extracts made from sea urchin embryos labeled with a [3H]RNA precursor.