title: Transcription of potato spindle tuber viroid by RNA polymerase II starts in the left terminal loop.
abstract: Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate autonomously in their host plants but do not code for a protein. Viroids of the family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type strain, are replicated by the host's DNA-dependent RNA polymerase II in the nucleus. To analyze the initiation site of transcription from the (+)-stranded circles into (-)-stranded replication intermediates, we used a nuclear extract from a non-infected cell culture of the host plant S. tuberosum. The (-)-strands, which were de novo-synthesized in the extract upon addition of circular (+)-PSTVd, were purified by affinity chromatography. This purification avoided contamination by host nucleic acids that had resulted in a misassignment of the start site in an earlier study. Primer-extension analysis of the de novo-synthesized (-)-strands revealed a single start site located in the hairpin loop of the left terminal region in circular PSTVd's secondary structure. This start site is supported further by analysis of the infectivity and replication behavior of site-directed mutants in planta.
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Geb. 26.12.U1, Universitätsstr.1, D-40225 Düsseldorf, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Kolonko
authors.forename: Oliver
authors.initials: O
authors.lastname: Bannach
authors.forename: Katja
authors.initials: K
authors.lastname: Aschermann
authors.forename: Kang-Hong
authors.initials: KH
authors.lastname: Hu
authors.forename: Michaela
authors.initials: M
authors.lastname: Moors
authors.forename: Michael
authors.initials: M
authors.lastname: Schmitz
authors.forename: Gerhard
authors.initials: G
authors.lastname: Steger
authors.forename: Detlev
authors.initials: D
authors.lastname: Riesner
mesh_terms: D010942:Plant Viruses / Q000235:genetics*; D012319:RNA Polymerase II / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; 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