IDENTIFICATION OF RECOGNITION ELEMENTS ON BACTERIOPHAGE-Q-BETA MINUS-STRAND RNA THAT ARE ESSENTIAL FOR TEMPLATE ACTIVITY WITH Q-BETA-REPLICASE

In order to identify the structural elements important for the activit y of the Q beta minus strand RNA as a template for Q beta replicase, a series of minus strand RNAs with internal or external deletions were prepared by in vitro transcription from suitable expression plasmids. The template activities of the deletion mutants were determined by sin gle-round replication assays using purified replicase holoenzyme or co re enzyme (lacking subunit alpha) in vitro. Two elements of RNA struct ure and/or sequence important for template activity were found. The fi rst is a segment in the 5'-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previou sly recognized to be highly conserved in the small variant MDV-1 RNA a nd suggested to be involved in its template recognition. The second el ement is defined by two partially complementary sequence segments in t he 5'-terminal region (map positions 557 to 576 and 24 to 35), that ap pear to be engaged in long-range base-pairing and may form the stem of a large secondary structure domain, whose branches are not necessary for template recognition. The results obtained with replicase holoenzy me and core enzyme mere identical within the accuracy of the method. T hey confirm the absence of any role of S1 protein in the interaction o f replicase with minus strand RNA and further emphasize the profound d ifference in the interactions of replicase with the plus and minus str and.