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Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation
T Baumstark1, A R Schröder, D Riesner
1Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
The EMBO Journal
|February 3, 1997
Summary
Potato spindle tuber viroid processing requires a specific RNA structure. A GNRA tetraloop-hairpin in the central conserved region is essential for correct viroid RNA processing in nuclear extracts.
Area of Science:
- Molecular Biology
- Virology
- RNA Structure
Background:
- Potato spindle tuber viroid (PSTVd) is a plant pathogenic RNA.
- Viroid RNA processing is a critical step in its replication cycle.
Purpose of the Study:
- To elucidate the structural requirements for the processing of PSTVd RNA.
- To identify the specific RNA motifs involved in PSTVd RNA cleavage and ligation.
Main Methods:
- S1 nuclease mapping and primer extension to identify cleavage sites.
- UV crosslinking, chemical mapping, and phylogenetic analysis to study RNA structure.
- Thermodynamic calculations to assess structural stability.
Main Results:
- A longer-than-unit-length PSTVd RNA transcript is processed in potato nuclear extract only when its central conserved region forms a multi-helix junction with at least one GNRA tetraloop-hairpin.
- The first cleavage occurs within the GNRA tetraloop stem, followed by a conformational change to a loop E motif.
- Unit-length linear intermediates are formed and subsequently ligated to mature circles.
Conclusions:
- The GNRA tetraloop-hairpin motif is crucial for PSTVd RNA processing.
- Specific structural rearrangements, including the transition from tetraloop to loop E, are key to the processing mechanism.
- Both autocatalytic and enzymatic ligation pathways contribute to the formation of mature circular viroid RNA.