Mapping in solution shows the peach latent mosaic viroid to possess a new pseudoknot in a complex, branched secondary structure

We have investigated the secondary structure of peach latent mosaic viroid (PLMVd) in solution, and we present here the first description of the struc ture of a branched viroid in solution, Different PLMVd transcripts of plus polarity were produced by using the circularly permuted RNA method and the exploitation of RNA internal secondary structure to position the 5' and 3' termini and studied by nuclease mapping and binding shift assays using DNA and RNA oligonucleotides, We show that PLMVd folds into a complex, branched secondary structure. In general, this structure is similar to that reporte d previously, which was based on sequence comparison and computer modelling . The structural microheterogeneity is apparently limited to only some smal l domains. More importantly, this structure includes a novel pseudoknot tha t is conserved in all PLMVd isolates and seems to allow folding into a very compact form. This pseudoknot is also found in chrysanthemum chlorotic mot tle viroid, suggesting that it is a unique feature of the viroid members of the PLMVd subgroup.