Formation of metastable RNA structures by sequential folding during transcription: Time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynam ically metastable structure containing a specific hairpin (HP II) in the (- )-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how P STVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure dis tribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by alt ering the concentration of nucleoside triphosphates to mimic the in vivo sy nthesis rate of DNA-dependent RNA polymerase II. With dependence on rate an d duration of the synthesis, the structure distributions were analyzed by t emperature-gradient gel electrophoresis (TGGE). Metastable structures are g enerated preferentially at low transcription rates-similar to in vivo rates -or at short transcription times at higher rates. Higher transcription rate s or longer transcription times lead to metastable structures in low or und etectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodyn amically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.