Inducible regulation of the S. cerevisiae cell cycle mediated by an RNA aptamer-ligand complex.
Previous studies have shown that the introduction of a ligand-binding RNA (aptamer) into the 5'-UTR of an mRNA can confer regulated expression of both prokaryotic and eukaryotic reporter genes. The current report shows that aptamer insertion into the 5'-UTR of a cyclin transcript in S. cerevisiae renders cell-cycle control dependent upon the presence or absence of the target ligand. A malachite green binding motif, defined by an asymmetric internal loop flanked by short RNA helices, was inserted immediately upstream of the CLB2 start codon. Progression through the cell cycle is dramatically slowed and elongated bud morphology develops when tetramethylrosamine (a fluorescent malachite green analogue) is added to the aptamer-containing strain. Quantification of CLB2 expression at the RNA and protein levels by RT-PCR and Western blot analysis, respectively, demonstrates that the aptamer ligand regulates transcript translatability rather than stability. One-dimensional NMR spectroscopy shows that the malachite green binding aptamer undergoes a dramatic ligand-dependent change in structure with many nucleotides folding to adopt a well-defined conformation. These results are consistent with a model in which translational initiation is blocked by ligand-induced conformational changes in the 5'-UTR.
Grate
D
D
Department of Biology and Center for the Molecular Biology of RNA, University of California at Santa Cruz, 95064, USA.
Wilson
C
C
D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology*; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006575:Heterocyclic Compounds, 3-Ring; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines; D012394:Rosaniline Dyes; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods; D014544:Uridine Triphosphate / Q000737:chemistry
C093749:CLB2 protein, S cerevisiae; D019926:Cyclin B; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D008024:Ligands; D009841:Oligonucleotides; D012333:RNA, Messenger; D012235:Rhodamines; D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins; C412715:tetramethylrosamine; C005095:malachite green; D014544:Uridine Triphosphate