Trinucleotide AUA and UAU formation catalyzed by wheat germ RNA polymerase II

Abstract

The elongation of dinucleotides ApU and UpA to trinucleotides ApUpA and UpApU by wheat germ RNA polymerase II was studied at a medium ionic strength (60 mM-KCl). The catalytic mechanism of the first internucleotide bond formation consists in the binding of the primer dinucleotide followed by the binding of NTP ("ordered bibi" reaction), i.e. by an analogous mechanism as found for RNA polymerase holoenzyme from E. coli. In further experiments phosphonate analogues of dinucleotides ApU and UpA were used as the priming dinucleotides. It was shown that analogues U(c)pA and Up(c)A are very poor primers for the synthesis of corresponding trinucleotides; the elongation of analogues A(c)pU and Ap(c)U was not observed at all. The comparison of kinetic constants Kia, KmA, KmB and Vmax as well as the substrate properties of phosphonate analogues indicates the increased specifity of the wheat germ RNA polymerase initiation binding site in comparison with the E. coli holoenzyme.