A Simple Method for Measuring of Ribonucleoside Triphosphates Specific Radioactivities using RNA Polymerase

Wc describe a method for the rapid, one-step determination of the specific radioactivity and pool size of ATP, UTP, CTP or GTP. Eschericlzia 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 [C]nucleoside triphosphate of known specific activity. The fact that [3H]nucleotide and ['4C]nucleotide are incorporated into poly[r(A-U)] or poly[r(G-C)] in equimolar amounts, coupled with a knowledge of the ['4C]nucleotide specific activity, permits calculation of the [3H]nucleotide specific activity. The requirement for direct knowledge of the ['4C]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. Rates of RNA synthesis and turnover can be determined by radioactive tracer methods only if the specific radioactivity of the immediate precursor ribonucleoside triphosphate is monitored during the experiment. The direct determination of specific radioactivities of ribonucleoside triphosphates has been relatively difficult. Column chromatographic methods are laborious and require large amounts of material. Sensitive enzymic methods such as the luciferase assay for ATP or GTP [l, 21 and the hexokinase assay for ATP or UTP [3] include time-consuming chromatographic steps. A recently described RNA polymerase method [4] permits determination of ATP or UTP pool size, but not specific radioactivity. This paper presents a rapid and sensitive method, requiring no chromatography, for the determination of the specific radioactivity and pool size of ATP, UTP, GTP or CTP. The method is based on the standard assay for Escherichia coli RNA polymerase [5].