and quenched with 10 volumes of stop solution at 0 C. Quantitation of primary transcripts and their cleavage prod- ucts was as detailed above, and the self-cleavage rate constants (at 2 mM MgCl2) were determined using the equa- tion derived previously (22). Values are the means of at least two independent experiments. RESULTS Some peculiarities of the hammerhead structures from ELVd The detailed morphology of both ELVd hammerheads has been reported previously (20) and, therefore, we will only Nucleic Acids Research, 2006, Vol. 34, No. 19 5615 summarize here their most salient features when compared with other natural hammerheads that have been used in this work. Both (+) and ( ) ELVd strands contain the 11 residues that form the central conserved core of natural hammerheads (3), ﬂanked by helix I/loop 1 (7 bp and 4 nt), helix II/loop 2 (4 bp and 4 nt forming a stable loop of the GNRA family) (23,24), and helix III (6 and 5 bp for + and hammerhead, respectively) (Figure 1 and Table 1). This morphology is similar to that of both hammerheads of PLMVd (25), and to the (+) hammerheads of CChMVd (26) and sTRSV (2) (Figure 1), although helices I of ELVd hammerheads are the longest reported in natural hammerheads (3), with that of the (+) hammerhead being particularly stable (5/7 bp are G–C). Figure 1. (A) Rod-like secondary structure that the nucleotides involved in both hammerheads adopt in the predicted most stable conformation of the reference variant of ELVd (+) RNA (accession number AJ536613). Sequences forming the hammerheads are delimited by flags, motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and ( ) polarities, respectively. (B) Structure of the ELVd (+) and ( ) hammerheads according to crystallographic data obtained for the Schistosoma mansoni hammerhead (16). Open square next to open triangle denotes Hoogsteen/sugar edge. Numbering of nucleotides and nomenclature of helices and loops is consistent with the standard criterion. Motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and ( ) polarities, respectively. The ovals represent the proposed tertiary interactions between loops 1 and 2 that increase the self-cleavage rates. The inset at the right displays the mutants derived from the ELVd (+) hammerhead used in the present work, with the changes introduced within boxes. (C) Structure of the (+) hammerheads of PLMVd, CChMVd and sTRSV.