title: A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs.
abstract: The internal ribosome site RNA of the cricket paralysis-like viruses (CrPV-like) binds directly to the ribosome, assembling the translation machinery without initiation factors. This mechanism does not require initiator tRNA, and translation starts from a non-AUG codon. A wealth of biochemical data has yielded a working model for this process, but the three-dimensional structure and biophysical characteristics of the unbound CrPV-like IRES RNAs are largely unexplored. Here, we demonstrate that the CrPV-like IRESes prefold into a two-part structure in the presence of magnesium ions. The largest part is a prefolded compact RNA domain that shares folding and structural characteristics with other compactly folded RNAs such as group I intron RNAs and RNase P RNA. Chemical probing reveals that the CrPV-like IRES' compact domain contains RNA helices that are packed tightly enough to exclude solvent, and analytical ultracentrifugation indicates a large change in the shape of the IRES upon folding. Formation of this compact domain is necessary for binding of the 40S subunit, and the structural organization of the unbound IRES RNA is consistent with the hypothesis that the IRES is functionally and structurally preorganized before ribosome binding.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Mail stop 8101, P.O. Box 6511, Aurora, CO 80045, USA.
authors.forename: David
authors.initials: D
authors.lastname: Costantino
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Kieft
mesh_terms: D019540:Area Under Curve; D001483:Base Sequence; D003001:Cloning, Molecular; D017665:Hydroxyl Radical; D008274:Magnesium / Q000378:metabolism; D015335:Molecular Probes; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes
chemical_list: D015335:Molecular Probes; D012367:RNA, Viral; D017665:Hydroxyl Radical; D008274:Magnesium