5618 Nucleic Acids Research, 2006, Vol. 34, No. 19 shown). Altogether these results indicate that the effects of changes in the trinucleotide preceding the self-cleavage site follow the same tendency observed previously in artiﬁcial trans-acting hammerheads under high magnesium concentra- tion (38–41). However, they neither provide hints on why some catalytically sub-optimal trinucleotides like AUA have been selected in natural hammerheads, nor on why the catalytically most favorable trinucleotide AUC has been essentially excluded in natural hammerheads. Co- and post-transcriptional self-cleavage may be different: a model for the lack of the trinucleotide AUC in most natural hammerheads Because RNA folding occurs during transcription (42), we considered the possibility that certain trinucleotides preced- ing the self-cleavage site, particularly AUC, might promote the transient adoption of catalytically-inactive metastable structures. If these metastable structures are indeed formed (see Discussion and Supplementary Figure S1), we reasoned that they could be indirectly revealed by their differential effect on self-cleavage very early during transcription, whereas the differences would be attenuated late in transcrip- tion when the most stable catalytically-active hammerhead outcompetes the metastable structures. The experimental data (Figure 4) are consistent with this model. Self-cleavage of the ELVd-(+)-AUC hammerhead during the initial tran- scription instants (15 s) was signiﬁcantly lower than that of the other three ELVd hammerheads (22 versus 46–67%), whereas later in transcription (1.5 min) the differences were less conspicuous (78 versus 89–91%), with self-cleavage being very similar for the four ELVd hammerheads (89– 94%) 10 min after initiating transcription (Figure 4A). Analo- gous results were obtained in independent experiments in which the magnesium concentration of the in vitro transcrip- tion mixture was, instead of 2 mM, 1 or 4 mM (data not shown). Following the approach for estimation of the self- cleavage rate constants during in vitro transcription (22), the values obtained for the four ELVd (+) hammerheads (at 2 mM magnesium and pH 8) were: ELVd-(+)-GUA> ELVd-(+)-AUA>ELVd-(+)-GUC>ELVd-(+)-AUC (Figure 4B). During in vivo transcription the differences could be even higher because interactions with host proteins forming part of the replication complex might stabilize the metastable structures, thus explaining why the trinucleotide AUC, the catalytically most favorable in a post-transcriptional context, is essentially absent in natural hammerheads. Comparative analysis with other natural hammerheads reveals two ELVd (+) hammerhead derivatives as the catalytically most active at low magnesium concentration To have a reliable estimate of the catalytic properties of the ELVd (+) hammerheads in relation to other known natural hammerheads, a self-cleavage kinetics analysis of the PLMVd (+), CChMVd (+) and sTRSV (+) hammerheads was performed at the same low magnesium concentrations. The morphology of these three hammerheads presents differ- ences in the helices and loops but the trinucleotide preceding their self-cleavage sites is GUC (Figure 1C). At 10 mM Figure 4. (A) Representative self-cleavage kinetics during transcription of the wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. Transcriptions (at pH 8 and 2 mM MgCl2) were made in the absence of the antisense oligodeoxyr- ibonucleotides. Reaction aliquots were removed at different time intervals, quenched with an excess of stop solution, separated by PAGE in 15% gels containing 8 M urea, and revealed and quantitated by a bioimage analyzer. Positions of the complete (C) primary transcript and of the resulting self- cleavage 30 fragment (30F), with their sizes in nt are indicated on the left. The smaller self-cleavage 50 fragment (9 nt) has migrated out of the gel. (B) Quantitative data were fitted to the equation derived previously (22) by a least square method. The inset displays the first 1.5 min for each experiment. magnesium, the self-cleavage rate constants of the PLMVd (+) and CChMVd (+) hammerheads were 5- to 6-fold lower than the comparable ELVd-(+)-GUC hammerhead (the ELVd variant with the same triplet preceding the self-cleavage site), whereas the self-cleavage rate constant of the sTRSV (+) hammerhead was negligible (Figure 5A). When compared with the fraction of self-cleaved molecules for the ELVd- (+)-GUC hammerhead at the end-point of the reaction (94%), the corresponding values for CChMVd (+) and, par- ticularly, for the PLMVd (+) hammerheads were signiﬁcantly lower (80 and 52%, respectively). At 50 mM magnesium