SPECIFIC SITE SELECTION IN RNA RESULTING FROM A COMBINATION OF NONSPECIFIC SECONDARY STRUCTURE AND -CCR- BOXES - INITIATION OF MINUS-STRANDSYNTHESIS BY TURNIP YELLOW MOSAIC-VIRUS RNA-DEPENDENT RNA-POLYMERASE

A turnip yellow mosaic Virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand syn thesis, which is initiated specifically opposite the 3'-CCA that termi nates the 3'-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstr eam of the pseudoknotted acceptor stem and 3'-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted dom ain could be functionally replaced by various simple stem/loops, altho ugh with some decrease in activity. The addition of varying numbers of consecutive -CCA- triplets to the 3' end of the tRNA-like structure r esulted in accurate initiation from each added triplet. A similar spec trum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA- triplets and no additional viral sequence. Substitu tion mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC- initiation s ite, but a preference for a purine immediately downstream. The introdu ction of secondary structure into the linear template showed that the usage of potential -CCR- initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the req uirement that a -CCR- sequence be sterically accessible. This mechanis m is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RN A virus replication and be applicable to other RNA-protein interaction s.