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. (C) 2001 Published by Elsevier Science Ltd.