Structural differences within the loop E motif imply alternative mechanisms of viroid processing.
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.
Owens
Robert A
RA
Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland 20705, USA. robert.a.owens@ars.usda.gov
Baumstark
Tilman
T
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010944:Plants / Q000821:virology; D012323:RNA Processing, Post-Transcriptional; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D013696:Temperature; D014466:Ultraviolet Rays; D014772:Viroids / Q000235:genetics* / Q000502:physiology* / Q000528:radiation effects; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D003432:Cross-Linking Reagents; D012367:RNA, Viral