In vitro Synthesis of RNA by Dissociated Lettuce Necrotic Yellows Virus Particles

The presence of an endogenous transcriptase in purified lettuce necrotic yellows virus (LNYV) particles has been confirmed. The enzyme requires a temperature of 22 °C, a pH value of 7.7, a detergent and Mg²⁺ concentration of 0.025% and 4 mM, respectively, for maximal synthesis of RNA; Zn²⁺, Ca²⁺ or Mn²⁺ inhibited the activity. The chelating agents ethylenediaminetetra-acetic acid (EDTA) and ethyleneglycolbis(2-aminoethylether)tetra-acetic acid (EGTA) stimulated the reaction, and in their presence the optimal Mg²⁺ concentration was higher. The in vitro transcription products of LNYV hybridized to the virus genome. After treatment of the virus with a non-ionic detergent the transcription complex could be separated by gradient centrifugation into two inactive fractions, the pellet and the upper part of the gradient. The activity could be restored by combining the two fractions.