Schröder Astrid R W, Riesner Detlev
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universtitätsstrasse 1, D-40225 Düsseldorf, Germany.
Nucleic Acids Res. 2002 Aug 1;30(15):3349-59. doi: 10.1093/nar/gkf454.
In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleotide was constructed which specifically binds to the HP II loop region in transient (-)-strand intermediates. Analysis of the resulting oligonucleotide/HP II complexes on temperature-gradient gels enabled us to follow the formation of HP II during in vitro transcription by T7 RNA polymerase. Moreover, we were able to demonstrate the formation of HP II during viroid replication in potato (Solanum tuberosum) cells.
在马铃薯纺锤块茎类病毒(PSTVd)的(-)链复制中间体中,一种含有特定发夹结构(HP II)的热力学亚稳结构被认为对类病毒复制至关重要。在本研究中,设计了一种方法,可使用生物物理方法在体外和体内直接检测HP II结构。构建了一种RNA寡核苷酸,它能特异性结合瞬时(-)链中间体中的HP II环区域。在温度梯度凝胶上对所得寡核苷酸/HP II复合物进行分析,使我们能够追踪T7 RNA聚合酶在体外转录过程中HP II的形成。此外,我们能够证明在马铃薯(Solanum tuberosum)细胞的类病毒复制过程中HP II的形成。