RNA-templated RNA replication is essential for viral or viroid infection, as 
well as for regulation of cellular gene expression. Specific RNA motifs likely 
regulate various aspects of this replication. Viroids of the Pospiviroidae 
family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in 
the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the 
role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. 
A tertiary-structural model of this motif, inferred by comparative sequence 
analysis and comparison with nuclear magnetic resonance and X-ray crystal 
structures of loop E motifs in other RNAs, is presented in which core 
non-Watson-Crick base pairs are precisely specified. Isostericity matrix 
analysis of these base pairs showed that the model accounts for the reported 
natural sequence variations and viable experimental mutations in loop E motifs 
of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us 
to design disruptive, as well as compensatory, mutations of PSTVd loop E. 
Functional analyses of such mutants by in vitro and in vivo experiments 
demonstrated that loop E structural integrity is crucial for replication, 
specifically during transcription. Our results suggest that the PSTVd loop E 
motif exists and functions in vivo and provide loss-of-function genetic evidence 
for the essential role of a viroid RNA three-dimensional motif in rolling-circle 
replication. The use of isostericity matrix analysis of non-Watson-Crick base 
pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro 
and in vivo functional assays of mutants offers a novel, comprehensive approach 
to elucidate the tertiary-structure-function relationships for RNA motifs of 
general biological significance.