Characteristics of a coupled cell-free transcription and translation system directed by vaccinia cores

Abstract

1. A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell-free system is based on a micrococcal-nuclease-treated reticulocyte lysate. 2. The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells. 3. Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores. 4. The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.