Published online 5 October 2006 Nucleic Acids Research, 2006, Vol. 34, No. 19 5613–5622 doi:10.1093/nar/gkl717 Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads Alberto Carbonell, Marcos De la Pen˜ a, Ricardo Flores* and Selma Gago Instituto de Biolog´ıa Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos, s/n, 46022 Valencia, Spain Received August 1, 2006; Revised September 15, 2006; Accepted September 16, 2006 ABSTRACT Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (1) and ( ) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (1) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd ( ) ham- merhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (1) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(1)-AUC ELVd-(1)-GUC.ELVd-(1)-GUA. ELVd-(1)-AUA. However, the ELVd-(1)-AUC ham- merhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive meta- stable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and pro- vide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(1)-GUC and ELVd-(1)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium. INTRODUCTION Hammerhead ribozymes were initially described in viroid and viroid-like satellite RNAs wherein they mediate self-cleavage of the multimeric RNA intermediates of one or both polarities resulting from replication through a rolling-circle mechanism [(1,2), for a review see Ref. (3)]. More speciﬁcally, hammer- head viroids follow a symmetric pathway of this mechanism in which the multimeric RNAs of both polarities self-cleave and subsequently are ligated into the corresponding mono- meric circular RNAs (4). Early structural dissection showed that minimized catalytically-active hammerheads have as few as 52 nt (5,6), igniting much interest in deciphering the catalytic mechanism (7–9) and developing its potential as a biotechnological tool (10). Most natural hammerheads are formed by a central conserved core ﬂanked by three double- stranded regions with relaxed sequence requirements (helices I, II and III), two of which (I and II) are capped by short loops (1 and 2, respectively). X-ray crystallography uncovered an intricate assortment of non-canonical interactions between the nucleotides of the central core, explaining their conserva- tion in natural hammerheads and illustrating that the actual contour does not resemble a hammerhead but rather a dis- torted Y in which the basal helix III is almost co-linear with helix II, whilst helix I forms an angle (7–9). However, these structural studies, which were performed with artiﬁcial hammerheads, are partially at odds with some biochemical data (11), and the use of artiﬁcial ribozymes designed to act in trans against speciﬁc RNA targets have met with limited success in vivo. More recent data have provided an explanation for these discrepancies by showing that modi- ﬁcations of loops 1 and 2 of natural hammerheads provoke a drastic reduction in their catalytic activity, indicating that these peripheral regions play a crucial role in catalysis through tertiary interactions between some of their nucleotides that may favor the active site at the low magnesium concentration *To whom correspondence should be addressed. Tel: +34 96 3877861; Fax: +34 96 3877859; Email: rflores@ibmcp.upv.es 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.