5620 Nucleic Acids Research, 2006, Vol. 34, No. 19 site may have an additional role in ligation. It is even possible that the higher rate and extent of ligation observed in a natural hammerhead with the interactions between loops 1 and 2 preserved could be relevant in physiological conditions (43), and that the trinucleotide preceding the self-cleavage/ ligation site may have some inﬂuence. Moreover, in natural contexts this trinucleotide might play functional roles other than in cleavage and ligation. This is illustrated by the case of CChMVd, in which the (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. A10 causes a moderate decrease of the trans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, but because A10 also occupies a singular and indispensable position in the global CChMVd conforma- tion as revealed by bioassays (44), these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. However, whereas the above arguments can explain why some catalytically sub-optimal trinucleotides have been selected in natural hammerheads, the reasons excluding the catalytically most favorable trinucleotide AUC in almost all natural hammerheads are probably different. We believe that this exclusion can be accounted by considering that self-cleavage during transcription and in a protein-free med- ium may be different. Following transcription of the ﬁrst hammerhead nucleotides, they can be kinetically trapped in a catalytically-inactive structure. These structures, predicted for the ELVd-(+)-AUC, ELVd-(+)-GUC, ELVd-(+)-GUA, and ELVd-(+)-AUA hammerheads with the Kinefold pro- gram (45) (Supplementary Figure S1), show that some trinu- cleotides preceding the self-cleavage site can potentially base pair with the CUGAU motif (hammerhead positions 3–7) (Figure 6). Moreover, the resulting interactions have different stability, with that formed by the trinucleotide AUC display- ing the highest and, as a consequence, delaying self-cleavage. This model also explains why when in natural hammerheads the ﬁrst nucleotide of the trinucleotide is A, the third must be also A (G is excluded because it would base pair with C3 and impede its role in the catalytic pocket, and U is also excluded because it can not form the complex array of non-canonical interactions) (11,16). Once transcription of the hammerhead is completed, the kinetic trap can be released as a result of the alternative interactions that can be formed (Figure 6), and the hammerhead can adopt its catalytically-active folding. Formation of the kinetic trap may be particularly facilitated in certain natural hammerheads, like the ELVd (+) hammerhead, with a long and stable helix I. It is Figure 6. Model for the transient folding of four hammerheads with different trinucleotides preceding the self-cleavage site. During transcription, nucleotides forming part of the hammerhead catalytic core (in dark background) can be transiently involved in catalytically-inactive metastable structures. The higher stability of the structure with the trinucleotide AUC preceding the self-cleavage site can specifically delay the adoption of the catalytically-active hammerhead. In the scheme of the catalytically-active hammerhead (right) the pair X–X represents C–G or U–A, and the Y represents C or A.