Poly(dAT) dependent trinucleotide synthesis catalysed by wheat germ RNA polymerase II. Effects of nucleotide substrates and cordycepin triphosphate.
Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT). Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts. The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase. Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis. The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur. During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway. The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme. Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA. However the ATP analogue cannot be incorporated into longer products than a trinucleotide. On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis. Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.