Different Mechanisms of Recognition of Bacteriophage Qβ Plus and Minus Strand RNAs by Qβ Replicase

Abstract

Our earlier work on the recognition of Qβ plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3′-end, i.e. the site of initiation of synthesis. Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity. In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP. A variant plus strand RNA containing a 490 nucleotide duplication near the 5′-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3′-end of the RNA. Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase. This suggests that the role of the host factor on the plus strand template is to bring the 3′-end into the proximity of the S-site/M-site domain, where replicase can initiate on it. In contrast, the 3′-end of the minus strand appears to be directly available to the enzyme.