Recognition of bacteriophage Qβ plus strand RNA as a template by Qβ replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor

Abstract

RNA-protein interactions between bacteriophage Qβ plus strand RNA and the components of the Qβ replicase system were studied by deletion analysis. Internal, 5′-terminal and 3′-terminal deletions were assayed for template activity with replicase in vitro. Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity. Binding complexes of the deletion mutant RNAs with the different proteins involved in Qβ RNA replication were analysed by electron microscopy. The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site. The same types of complexes observed with replicase were also formed with purified protein S1 (the α subunit of replicase), suggesting that these internal interactions with Qβ RNA are mediated by the S1 protein. The Qβ host factor, a protein required for the template activity of the Qβ plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3′-end, resulting in double-looped structures. The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites. An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs. Qβ RNA molecules with 3′-truncations formed 3′-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3′-end by host factor is not dependent on a specific 3′-terminal base sequence.