Nucleic Acids Research, 2006, Vol. 34, No. 19 5619 Figure 5. Representative self-cleavage kinetics at 10 mM (A) and 50 mM (B) magnesium of the purified ELVd-(+)-GUC mutant hammerhead and of three other natural hammerheads with the trinucleotide GUC preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft ¼ F1(1 e kt), where F1 is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F1 (between parenthesis) are shown below. ND, not detectable self-cleavage. The concentration of Mg was 1000-fold higher than that of hammerheads. the differences became somewhat diminished but the rate constant and the extent of self-cleavage of the ELVd-(+)- GUC hammerhead were still the highest (Figure 5B). There- fore, the latter hammerhead, and the ELVd-(+)-AUC mutant (Figure 3), appear as the catalytically most active in this particular post-transcriptional context. DISCUSSION The study of hammerheads in their cis context and at the low magnesium concentration existing in vivo has still received limited interest, despite its implications for a better under- standing of the catalytic mechanism and biological diversity of natural hammerheads, as well as for the development of artiﬁcial trans-acting hammerheads with increased activity in physiological conditions. Here we have examined the self- cleavage kinetics at low magnesium concentration of the hammerheads described recently in ELVd (20) and, from the effects of changes in the trinucleotide preceding the self-cleavage site, we have tried to derive some clues about the rules governing the limited variability of this trinucleotide in natural hammerheads. Starting from the ELVd (+) hammerhead, with an AUA trinucleotide preceding the self-cleavage site, we changed this trinucleotide into GUC. Due to the very similar morphol- ogy of both ELVd hammerheads (Figure 1B), this substitu- tion transformed the ELVd (+) hammerhead in a derivative analogous to the ELVd ( ) hammerhead. Kinetic analysis at two low magnesium concentrations (10 and 50 mM) showed that the self-cleavage rate constant of the ELVd- (+)-GUC mutant was considerably higher than that of ELVd-(+)-AUA wild-type (Figure 3), raising the question of why two trinucleotides have been differentially selected in the two ELVd hammerheads. Moreover, under the same magnesium concentrations, the self-cleavage rate constants of the ELVd-(+)-AUC and ELVd-(+)-GUA mutants were similar to and somewhat lower than those of the ELVd-(+)-GUC mutant, respectively. Whereas the GUA trinucleotide precedes the self-cleavage site of two natural hammerheads (Table 1), why the catalytically very favorable trinucleotide AUC has been excluded in most natural hammerheads? The effect of the trinucleotide preceding the self-cleavage site on the rate constant has been previously studied in sev- eral artiﬁcial trans-acting hammerheads at high magnesium concentration. Although there are some discrepancies that probably reﬂect the distinct characteristics of speciﬁc hammerheads, the highest self-cleavage rate constants were observed for the trinucleotides GUC and AUC, whereas those for the trinucleotides GUA and AUA were signiﬁcantly smaller (38–41). These results parallel those reported here at low magnesium concentration with the cis-acting ELVd (+) hammerhead and its mutants, indicating that they are to a good extent independent of the cis or trans context. In the framework of ELVd replication, the two trinucleo- tides preceding the self-cleavage site may have been differen- tially selected in the two hammerheads because of the need of differential self-cleavage rates during transcription of (+) and ( ) strands. Replication additionally entails subsequent ligation of the self-cleaved RNAs. It is not yet clear whether in hammerhead viroids of the family Avsunviroidae this is an enzyme-catalyzed reaction or it occurs non-enzymatically (self-ligation) giving rise to a 20,50-phosphodiester bond [see (4) for a Discussion], but the RNA structures promoting self-cleavage and ligation may well be not be the same and, consequently, the trinucleotides preceding the self-cleavage