A BRANCHED STEM-LOOP STRUCTURE IN THE M-SITE OF BACTERIOPHAGE-Q-BETA RNA IS IMPORTANT FOR TEMPLATE RECOGNITION BY Q-BETA REPLICASE HOLOENZYME

An internal site on bacteriophage Q beta RNA, the M-site (map position 2545 to 2867), was recently shown by us to be required for the effici ent initiation of minus strand synthesis by Q beta replicase. In a mor e detailed mutational analysis, we show here that the essential elemen ts within the M-site consist of two successive stem-loop structures fo llowed by a bulge loop of unpaired purines, located at nucleotides 269 6 to 2754 on the tip of a long, imperfectly base-paired stalk. Mutatio nal changes affecting the sequences of paired or unpaired nucleotides in this segment reduced the template efficiency only mildly. The only severe effects were observed when one of the helical stems or the unpa ired bulge was completely deleted or substantially shortened. We concl ude that the three-dimensional backbone arrangement of these three ele ments constitutes the feature recognized by replicase. The role of the long stalk remains undetermined, because mutations that either stabil ized or disrupted its base-pairing barely affected template activity, and even deletion of a major portion of one of its strands did not cau se complete inactivation. Earlier evidence had implicated protein S1 ( the alpha subunit of replicase) as the mediator of the M-site interact ion. The lack of an active M-site on the Q beta RNA template has the s ame quantitative and qualitative effects on template recognition as th e absence of the S1 protein from replicase in the presence of wild-typ e RNA. We therefore believe that the M-site interaction explains most of the role of S1 protein in the replication of Q beta RNA by replicas e. (C) 1998 Academic Press.