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- Repsilber, Dirk, 1971-, et al.
(författare)
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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
- 1999
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Ingår i: RNA. - New York, USA : Cambridge University Press. - 1355-8382 .- 1469-9001. ; 5:4, s. 574-84
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Tidskriftsartikel (refereegranskat)abstract
- A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically 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 PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically 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.
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