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Structural requirements for viroid processing by RNase T1.

G Steger1, T Baumstark, M Mörchen

  • 1Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, F.R.G.

Journal of Molecular Biology
|October 5, 1992
PubMed
Summary
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Viroids are processed by RNase T1 through a specific secondary structure that facilitates cleavage and ligation. This mechanism, observed in vitro, provides a model for cellular viroid replication.

Area of Science:

  • Plant pathology
  • Molecular biology
  • Virology

Background:

  • Viroid replication involves enzymatic cleavage and ligation of oligomeric (+) strand intermediates.
  • Potato spindle tuber viroid (PSTVd) transcripts can be processed into circular forms by RNase T1 in vitro.

Purpose of the Study:

  • To elucidate the processing site and mechanism of PSTVd transcripts by RNase T1.
  • To investigate the role of secondary structure in viroid processing.

Main Methods:

  • Construction and analysis of 16 site-specific mutants of a longer-than-unit-length PSTVd transcript.
  • In vitro processing assays with RNase T1.
  • Infectivity studies.
  • Temperature-gradient gel electrophoresis.
  • Secondary structure calculations.

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Main Results:

  • Wild-type and some mutated transcripts adopt a specific "processing structure" essential for RNase T1 cleavage and ligation.
  • This structure spatially juxtaposes cleavage sites, promoting ligation to circular molecules.
  • Mutants unable to form this structure were not processed.
  • The in vitro processing mechanism mirrors cellular processing.

Conclusions:

  • Viroid structural features enable simultaneous cleavage and ligation by RNase T1.
  • The identified "processing structure" is crucial for efficient in vitro and potentially in vivo processing.
  • This study offers a mechanistic model for cellular viroid processing.