title: Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.
abstract: 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.
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Fels
authors.forename: K
authors.initials: K
authors.lastname: Hu
authors.forename: D
authors.initials: D
authors.lastname: Riesner
mesh_terms: D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics*; D012319:RNA Polymerase II / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000821:virology; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics*; D014772:Viroids / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D012319:RNA Polymerase II; D004720:Endonucleases