Internal and 3' RNA initiation by Qβ replicase directed by CCA boxes

Abstract

RNA initiation by Qβ replicase directed by the short-sequence CCA at the 3'-end of all RNAs amplified by this enzyme has been studied. Most CCA repeats in an RNA consisting of 12 CCAs serve as independent sites of de novo RNA initiation, with initiation occurring opposite the 3'-C residue of each CCA. Qβ replicase is thus capable of internal initiation remote from the 3'- end, although predominant initiation occurs close to the 3'-end. The precise 3'-terminal sequence in (CCA)(n)-containing RNAs influences the number and position of active initiation sites near the 3'-terminus. C residues are required at the initiation site, whereas the position of purines (especially A residues) influences the selection of initiation sites. The template activity of (CCA)(n) RNAs is positively correlated with the number of CCA repeats. Three CCA repeats added to the 3'-end of a nontemplate 83-nt RNA are sufficient to activate transcription by Qβ replicase. These experiments show that CCA boxes can act as strong initiation sites in the absence of specific cis-acting signals derived from Qβ RNA, although the efficiency of initiation is modulated by surrounding sequence. (C) 2000 Academic Press.