Nucleic Acids Research

KTnetics of RNA chain elongation catalyzed by wheat germ RNA polymerase II have been studied using various synthetic DNA templates in the presence of excess dinucleotide monophosphate primers. With singleor double-stranded homopolymer templates, the double reciprocal plots l/(velocity) as a function of l/(nucleotide substrate) exhibit positive, negative or no curvature. With poly(dAT) as template, the mechanism of nucleoside monophosphate incorporation into RNA is not the ping-pong kinetic mechanism which was derived for E.coli RNA polymerase (6). Noncomplementary nucleoside triphosphates inhibit RNA transcription allosterically. Cordycepin triphosphate behaves as ATP, and not only inhibits AMP incorporation but also that of UMP and GMP on appropriate templates. The reason for this complex kinetic behavior is not yet understood. Possibilities are raised that there are several nucleoside triphosphate binding sites on wheat germ RNA polymerase II, that additional nucleoside triphosphate dependent enzymatic activities are required for reaction to occur or that the Km value for incorporation of a given nucleoside monophosphate into RNA is dependent on the length of the RNA chain and/or the nucleotide sequence surrounding the complementary base on the DNA template. INTRODUCTION The basic features of the transcription reaction can be summarized as (reviewed in 1): (i) enzyme binding to DNA template and localized melting of the helix; (ii) initiation corresponding to the formation of the first phosphodiester bond: a purine nucleoside triphosphate at the 5' end of the RNA synthesized is usually required; (iii) elongation involving the sequential incorporation of nucleoside monophosphates; and (iv) termination of the RNA chain. It is generally felt that the control of RNA synthesis in eucaryotic cells probably lies in the initial steps of transcription (RNA polymerase: DNA recognition process, initiation of RNA synthesis) rather than in other steps of the polymerization reaction. However, recent results illustrate the possible importance of other steps in eucaryotic transcription. Thus Dauphinais (2) suggested that, rDNA transcription during lymphocyte activation might be controlled at the level of the elongation reaction. Studies conducted with plant RNA polymerases II also suggest that the elongation-translocation © IRL Press Limited, Oxford, England. 3303 Nucleic Acids Research properties of the enzymes could depend on the nucleotide sequence (and/or conformation) of the DNA sequence being transcribed. Thus pausing and non processivity of plant RNA polymerases II in RNA synthesis have been observed with both natural (3) and synthetic DNA templates (4). Similar observations have been reported during the in vitro transcription of the yeast alcohol deshydrogenase I gene by yeast RNA polymerase II (5). Relatively little is known, however, about the actual mechanism of enzymatic polymerization catalyzed by eucaryotic RNA polymerases. For E. coli RNA polymerase, for which the results are best documented, Rhodes and Chamberlin (6) proposed that the enzyme is kinetically characterized in the elongation step by a single binding site for the nucleoside triphosphate substrates. By applying steady-state kinetics, these authors have determined the Ks values for the nucleotide substrates on a number of synthetic templates. In their studies, differences in the Ks values were not large, and did not depend on the DNA base sequence. Low efficiency competitive inhibition of the elongation reaction is observed with high concentrations of noncomplementary nucleotides, which is attributed to a general affinity of the polymerase in the enzyme/RNA/DNA ternary complex for nucleoside triphosphates. Therefore, all ternary complexes have equal affinity for noncomplementary nucleoside triphosphates. From the kinetic study using these complexes, a simple ping-pong kinetic model was derived and was shown to fit the data obtained with alternating copolymer templates (6). Although information concerning the mechanism of interaction of eucaryotic RNA polymerases with nucleoside triphosphates is scarse, recent results on RNA polymerases from higher plant cells (soybean, parsley and wheat germ) revealed that these enzymes could be allosterically regulated (7-9). These studies suggested that the enzymes contain from two to five ligand sites. In addition, Grossmann and Seitz (7-8) showed that nucleoside triphosphates in excess of the divalent cations acted as allosteric inhibitors of enzyme activity. These few results tend to support the contention that nucleoside triphosphates and divalent cations may act as low molecular weight regulators of transcription in eucaryotic cells. In this context, these interesting properties may reveal hiterto unconsidered mechanisms for regulation of transcription. From the above concepts, and in view of the fundamental importance of nucleoside triphosphates in controlling the activity of RNA polymerases, we have undertaken a kinetic study of the RNA chain elongation reaction catalyzed by wheat germ RNA polymerase II. In this initial study, the reaction mechanism was investigated by means of steady-state kinetics. The results obtained using various synthetic templates are compared to those reported for E. coli RNA polymerase (see for instance 1 and 10).