Characteristics of a Coupled Cell‐Free Transcription and Translation System Directed by Vaccinia Cores

Abstract

A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesized in situ by vaccinia virus cores. The cell-free system is based on a micrococcal-nuclease-treated reticulocyte lysate. The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected [rabbit kidney and hamster kidney] cells. 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 ds[double-stranded]RNA associated with the cores. The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation 10-fold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.