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

RNA-protein interactions between bacteriophage Q beta plus strand RNA and the components of the Q beta 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 Q beta 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 alpha subunit of replicase), suggesting that these internal interactions with Q beta RNA are mediated by the S1 protein. The Q beta host factor, a protein required for the template activity of the Q beta 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 pat terns 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. Q beta 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. (C) 1997 Academic Press Limited.