In vitro and in vivo studies of the RNA conformational switch in Alfalfa mosaic virus.
The 3' termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3' untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3' UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3' UTR of AMV RNAs.
Chen
Shih-Cheng
SC
Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, Einsteinweg 55, 2300 RA, Leiden, The Netherlands.
Olsthoorn
René C L
RC
D020413:3' Untranslated Regions; D017794:Alfalfa mosaic virus / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D017931:DNA Primers; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D014779:Virus Replication
D020413:3' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral