Resynchronization of RNA synthesis by coliphage Qbeta replicase at an internal site of the RNA template

In previous work Qβ replicase has been used to synthesize labelled 5′ terminal segments of Qβ plus or minus strands of defined length. A procedure has now been developed which allows resynchronization of Qβ replicase at an internal position and synthesis of a labelled minus-strand segment complementary to the coat cistron ribosome binding site and the intercistronic region between the A₂ (maturation) and the coat cistron. Resynchronization is accomplished by binding a ribosome to Qβ RNA and allowing Qβ replicase to initiate and elongate up to the ribosome, using unlabelled ribonucleoside triphosphates. The ribosome is dissociated by EDTA treatment and the EDTA is removed. The replicating complex remains functional after this treatment, and addition of labelled substrates leads to synchronized elongation. The radioactive part of the product recovered after a short elongation period with labelled substrates was shown to be complementary to the coat protein ribosome binding site.