also interesting to note that, in nature, an AUC trinucleotide preceding the self-cleavage site has been only found in the Schistosome douthitti hammerhead, whereas this trinucleotide is GUC in the other Schistosome spp. hammerheads deposited in databases. Due to the particular morphology of this hammerhead, adoption of a catalytically-inactive metastable structure cannot be facilitated by helix I because one of its strands is transcribed subsequently to all the other sequences forming the hammerhead. In summary, co- and post-transcriptional self-cleavage, particularly in vivo, may be different, with natural hammerheads having been probably selected through evolution to function under conditions for co-transcriptional self-cleavage. Hence, a deeper understand- ing of hammerheads (and probably other ribozymes) in their natural RNA context requires kinetic analysis not only in the cis-cleaving format but also during transcription. Pertinent to this point is the ﬁnding that transcription initiation of viroid strands occurs at speciﬁc sites, the location of which in the nascent strands is consistent with avoiding structures that dis- favor the adoption of the catalytically-active hammerheads (46,47). Finally, the kinetics analysis here reported also shows that the ELVd-(+)-GUC hammerhead presents the highest rate constant and extent of self-cleavage (in low magnesium and in a post-transcriptional context) when compared with three other natural hammerheads from two viroids and one satellite RNA in which the trinucleotide preceding the self-cleavage site is also GUC. This may be the result of the high thermo- dynamic stability of the helices of the ELVd-(+)-GUC ham- merhead that would facilitate the tertiary interactions between the peripheral regions important for catalysis. Therefore, this hammerhead, and the ELVd-(+)-AUC mutant, can serve as a base for designing new and efﬁcient trans-acting hammer- heads in which the tertiary interactions are preserved. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS This work has been partially supported by grants BFU2005- 06808/BMC from the Ministerio de Educacio´ n y Ciencia (MEC) and ACOMP06/141 from the Generalidad Valenciana (Spain). A.C. has been supported by a pre-doctoral fellowship from the MEC, M.D.P. by a long-term postdoctoral fellow- ship of the International Human Frontier Science Program Organization and by a ‘Ramo´ n y Cajal’ contract from the MEC, and S.G. by a postdoctoral fellowship from the Fundaci o´n ‘Jose´ y Ana Royo’ and by an I3P contract from the Consejo Superior de Investigaciones Cient´ıficas. Funding to pay the Open Access publication charges for this article was provided by the MEC. Conflict of interest statement. None declared. REFERENCES 1. Hutchins,C.J., Rathjen,P.D., Forster,A.C. and Symons,R.H. (1986) Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res., 14, 3627–3640. Nucleic Acids Research, 2006, Vol. 34, No. 19 5621 2. Prody,G.A., Bakos,J.T., Buzayan,J.M., Schneider,I.R. and Bruening,G. (1986) Autolytic processing of dimeric plant virus satellite RNA. Science, 231, 1577–1580. 3. Flores,R., Herna´ndez,C., De la Pen˜a,M., Vera,A. and Daro`s,J.A. (2001) Hammerhead ribozyme structure and function in plant RNA replication. Methods Enzymol., 341, 540–552. 4. Flores,R., Herna´ndez,C., Mart´ınez de Alba,E., Daro`s,J.A. and Di Serio,F. (2005) Viroids and viroid-host interactions. Ann. Rev. Phytopathol., 43, 117–139. 5. Forster,A.C. and Symons,R.H. (1987) Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell, 49, 211–220. 6. Forster,A.C. and Symons,R.H. (1987) Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell, 50, 9–16. 7. Pley,H.W., Flaherty,K.M. and McKay,D.B. (1994) Three-dimensional structure of a hammerhead ribozyme. Nature, 372, 68–74. 8. Scott,W.G., Finch,J.T. and Klug,A. (1995) The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell, 81, 991–1002. 9. Scott,W.G., Murray,J.B., Arnold,J.R., Stoddard,B.L. and Klug,A. (1996) Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science, 274, 2065–2069. 10. Rossi,J.J. and Couture,L.A. (eds) (1999) Intracellular Ribozyme Applications: Principles and Protocols. Horizon Scientific Press, Wymondham, UK. 11. Blount,K.F. and Uhlenbeck,O.C. (2005) The structure-function dilemma of the hammerhead ribozyme. Annu. Rev. Biophys. Biomol. Struct., 34, 415–440. 12. De la Pen˜a,M., Gago,S. and Flores,R. (2003) Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity. EMBO J., 22, 5561–5570. 13. Khvorova,A., Lescoute,A., Westhof,E. and Jayasena,S.D. (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nature Struct. Biol., 10, 708–712. 14. Canny,M.D., Jucker,F.M., Kellogg,E., Khvorova,A., Jayasena,S.D. and Pardi,A. (2004) Fast cleavage kinetics of a natural hammerhead ribozyme. J. Am. Chem. Soc., 126, 10848–10849. 15. Penedo,J.C., Wilson,T.J., Jayasena,S.D., Khvorova,A. and Lilley,D.M. (2004) Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements. RNA, 10, 880–888. 16. Martick,M. and Scott,W.G. (2006) Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell, 126, 1–12. 17. Saksmerprome,V., Roychowdhury-Saha,M., Jayasena,S., Khvorova,A. and Burke,D.H. (2004) Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures. RNA, 12, 1916–1924. 18. Weinberg,M.S. and Rossi,J.J. (2005) Comparative single-turnover kinetic analyses of trans-cleaving hammerhead ribozymes with naturally derived non-conserved sequence motifs. FEBS Lett., 579, 1619–1624. 19. Burke,D.H. and Greathouse,S.T. (2005) Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities. BMC Biochem., 6, 14. 20. Fadda,Z., Daro`s,J.A., Fagoaga,C., Flores,R. and Duran-Vila,N. (2003) Eggplant latent viroid (ELVd): candidate type species for a new genus within family Avsunviroidae (hammerhead viroids). J. Virol., 77, 6528–6532. 21. Flores,R., Daro`s,J.A. and Herna´ndez,C. (2000) The Avsunviroidae family: viroids with hammerhead ribozymes. Adv. Virus Res., 55, 271–323. 22. Long,D.M. and Uhlenbeck,O.C. (1994) Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. Proc. Natl Acad. Sci. USA, 91, 6977–6981. 23. Woese,C.R., Winker,S. and Gutell,R.R. (1990) Architecture of ribosomal RNA: constraints on the sequence of ‘tetra-loops’. Proc. Natl Acad. Sci. USA, 87, 8467–8471. 24. Heus,H.A. and Pardi,A. (1991) Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science, 253, 191–194. 25. Herna´ndez,C. and Flores,R. (1992) Plus and minus RNAs of peach latent mosaic viroid self cleave in vitro via hammerhead structures. Proc. Natl Acad. Sci. USA, 89, 3711–3715.