A long-range interaction in Q_β RNA that bridges the thousand nucleotides between the M-site and the 3′ end is required for replication

Abstract

The genome of the positive strand RNA bacteriophage Q_β folds into a number of structural domains, defined by long-distance interactions. The RNA within each domain is ordered in arrays of three- and four-way junctions that confer rigidity to the chain. One such domain, RD2, is about 1,000-nt long and covers most of the replicase gene. Its downstream border is the 3′ untranslated region, whereas upstream the major binding site for Q_β replicase, the M-site, is located. Replication of Q_β RNA has always been puzzling because the binding site for the enzyme lies some 1,500-nt away from the 3′ terminus. We present evidence that the long-range interaction defining RD2 exists and positions the 3′ terminus in the vicinity of the replicase binding site. The model is based on several observations. First, mutations destabilizing the long-range interaction are virtually lethal to the phage, whereas base pair substitutions have little effect. Secondly, in vitro analysis shows that destabilizing the long-range pairing abolishes replication of the plus strand. Thirdly, passaging of nearly inactive mutant phages results in the selection of second-site suppressor mutations that restore both long-range base pairing and replication. The data are interpreted to mean that the 3D organization of this part of Q_β RNA is essential to its replication. We propose that, when replicase is bound to the internal recognition site, the 3′ terminus of the template is juxtaposed to the enzyme's active site.