Evidence for allosterism in in vitro DNA synthesis on RNA templates

Abstract

Hemoglobin mRNA and (rA)(n).(dT)(10) have been used as primer-templates in a kinetic study of DNA synthesis with Escherichia coli DNA polymerase I (DNA nucleotidyl transferase, EC 2.7.7.7) and Mason-Pfizer monkey virus reverse transcriptase (RNA-directed DNA polymerase). The rate versus enzyme concentration curve is sigmoidal and is consistent with a cooperative phenomenon. The results could be interpreted in terms of the formation of an active complex containing enzyme dimers (or oligomers) on the primer-template. We have also observed sigmoidal kinetics in rate versus deoxynucleotide triphosphate concentration. These results are consistent with an allosteric mechanism in which the triphosphates act as both modifiers and DNA precursors. In the critical range, a 6- to 8-fold increase in both enzyme and triphosphate concentrations can lead to a 1500-fold increase in the rate of synthesis on an RNA template. Thus, small changes in enzyme and precursor concentrations could play a regulatory role in vivo.