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Nucleic Acids Research Advance Access published online on November 29, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn918
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© 2008 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.

RNA

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

David Dufour1, Marcos de la Peña2, Selma Gago2, Ricardo Flores2,* and José Gallego1,*

1Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46012 Valencia and 2Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avda. de los Naranjos s/n, 46022 Valencia, Spain

*To whom correspondence should be addressed. Tel: +34 963 289680; Fax: +34 963 289701; Email: jgallego{at}cipf.es

*Correspondence may also be addressed to Ricardo Flores. Tel: +34 963 877861; Fax: +34 963 877859; Email: rflores{at}ibmcp.upv.es

Received September 12, 2008. Revised October 27, 2008. Accepted October 30, 2008.

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5' side and an extrahelical U at its 3'-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3'-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5' U of loop 1 and the 3' A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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