Over the past two decades, many studies have reported on the
inhibition of the HHR by variable metabolites such as aminoglycoside
antibiotics [11], terbium (III) [12], and cobalt hexamine [13], in vitro as
well as in vivo [14], but none of the ribozymes used in these studies
was from a hammerhead viroid. In addition, most of these ribozymes
were minimal trans-acting constructs although the physiological reaction
is cis-acting. Furthermore, this reaction involves the native peripheral
regions of the ribozyme [15,16] which, by interacting with each other,
facilitate and stabilize folding into a single active structure. These
regions are necessary for optimal activity in physiological conditions,
although they are not directly involved in the catalysis [17].
Consequently, the present investigation made use of the HHR of the
Chrysanthemum chlorotic mottle viroid (CChMVd). The structure and
function of the peripheral loops of this ribozyme were previously studied using a combination of NMR spectroscopy, site-direct mutagenesis,
kinetic studies and infectivity analyses [18]. The results obtained
provided insights into the three-dimensional folding of the HHR and
emphasize the importance of almost all the nucleotides in the terminal
loops for self-cleavage of the ribozyme in vitro and for infectivity of the
viroid in vivo. Recently, our laboratory investigated the cis-cleavage
reaction of a CChMVd-HHR (Fig. 1) by the high pressure approach on
the over-all cleavage reaction [19]. Two different conformations of
active molecules were identified in the reaction mixture corresponding
to fast and slow cleaving ribozymes.