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 a ptamer insertion into the 5'-UTR of a cyclin transcript in S. cerevisiae re nders cell-cycle control dependent upon the presence or absence of the targ et ligand. A malachite green binding motif, defined by an asymmetric intern al loop flanked by short RNA helices, was inserted immediately upstream of the CLB2 start codon. Progression through the cell cycle is dramatically sl owed and elongated bud morphology develops when tetramethylrosamine (a fluo rescent 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 lig and regulates transcript translatability rather than stability. One-dimensi onal NMR spectroscopy shows that the malachite green binding aptamer underg oes a dramatic ligand-dependent change in structure with many nucleotides f olding to adopt a well-defined conformation. These results are consistent w ith a model in which translational initiation is blocked by ligand-induced conformational changes in the 5'-UTR. (C) 2001 Published by Elsevier Scienc e Ltd.