Cell-free protein synthesis resulting in active phage Qbeta replicase.

Abstract

SEVERAL steps in the replication of the small phage Qβ can be reconstructed under cell-free conditions. Net synthesis of biologically active RNA “plus” strands has been achieved in vitro using RNA from virus particles as a template and RNA dependent RNA polymerase or “replicase” from infected E. coli cells1. This reaction proceeded through complementary RNA or “minus” strands as intermediate templates2. The plus strand-dependent reaction required a “host factor” HF I in addition to purified replicase3–6,7. Furthermore, three of four phage coded proteins can be synthesized in vitro using Qβ RNA as an mRNA and an extract from uninfected E. coli as a protein synthesizing system8,9. Coat protein and the phage specific subunit of replicase (R protein) when synthesized in vitro became bound to Qβ RNA8,10 as would be expected for the authentic proteins. Cell-free synthesis of enzymatically active Qβ replicase, however, has not been reported so far.