Identification of Recognition Elements on Bacteriophage Qβ Minus Strand RNA that are Essential for Template Activity with Qβ Replicase

Abstract

In order to identify the structural elements important for the activity of the Qβ: minus strand RNA as a template for Qβ: 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 single-round replication assays using purified replicase holoenzyme or core enzyme (lacking subunit α) in vitro. Two elements of RNA structure and/or sequence important for template activity were found. The first is a segment in the 5′-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previously recognized to be highly conserved in the small variant MDV-I RNA and suggested to be involved in its template recognition. The second element is defined by two partially complementary sequence segments in the 3′-terminal region (map positions 557 to 576 and 24 to 35), that appear 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 holoenzyme and core enzyme were identical within the accuracy of the method. They confirm the absence of any role of S1 protein in the interaction of replicase with minus strand RNA and further emphasize the profound difference in the interactions of replicase with the plus and minus strand.