Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites

Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (–)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by α-amanitin. RT–PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A111 and A325. The sequences of the first 7 nt transcribed are very similar, 105GGAGCGA111 and 319GGGGCGA325. GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (–)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties.