Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires 
extensive rearrangement of the highly base-paired native structure. For Potato 
spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by 
the change from a multibranched tetraloop structure to a loop E conformation. 
Here we present evidence that processing of Citrus viroid III (CVd-III), a 
member of a related group of viroids that also replicate in the nucleus, may 
proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs 
with DMS and CMCT revealed that the loop E motifs of these two viroids have 
quite different tertiary structures. As shown by temperature gradient gel 
electrophoresis, the presence of two likely Watson-Crick GC pairs results in a 
significant overall stabilization of the CVd-III loop E-like motif. Unlike 
PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA 
tetraloop, and comparison of suboptimal structures indicates that the initial 
cleavage event could occur on the 5' side of the only GU wobble pair in a helix 
involving a nearby pair of inverted repeats. According to our model, 
rearrangement of 3' sequences into a hairpin stem containing an identical 
arrangement of GC, GU, and CG base pairs and a second cleavage event is followed 
by formation of loop E, which serves to align the 5' and 3' termini of the 
CVd-III monomer prior to ligation. Because ligation would occur within loop E 
itself, stabilization of this motif may be needed to hold the 5' and 3' termini 
of CVd-III in position for the host ligase.