In:
RNA, Cold Spring Harbor Laboratory, Vol. 12, No. 10 ( 2006-10), p. 1868-1882
Abstract:
During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5 Δ p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5 Δ p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5 Δ p can bind to a single stem–loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5–RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5′ end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3′ end of the molecule, including the conserved ACA box.
Type of Medium:
Online Resource
ISSN:
1355-8382
,
1469-9001
Language:
English
Publisher:
Cold Spring Harbor Laboratory
Publication Date:
2006
detail.hit.zdb_id:
1475737-0
SSG:
12
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