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 alpha-amanitin. RT-PCR revealed the existence of at least two 
different start sites and primer extension identified these as nucleotides 
A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, 
(105)GGAGCGA(111) and (319)GGGGCGA(325). 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.