Tetracycline‐aptamer‐mediated translational regulation in yeast

Abstract

SummaryWe describe post-transcriptional gene regulation in yeast based on direct RNA-ligand interaction. Tetracycline-dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5 ¢ ¢ ¢ ¢ untranslated region (5 ¢ ¢ ¢ ¢ UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap-distal) in the 5 ¢ ¢ ¢ ¢ UTR was more effective than repression via a capproximal position. On the other hand, suppression in a cell-free system reached maximally 50-fold and was most effective via a cap-proximal aptamer. Examination of the kinetics of tetracycline-dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell-extracts. Tetracycline-dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap-proximal insertion inhibits binding of the 43S complex to the cap structure whereas start-codon-proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.

Screening for engineered neomycin riboswitches that control translation initiation

“…However, there are only a few aptamers that have been reported that act as a riboswitch in vivo (Werstuck and Green 1998;Grate and Wilson 2001;Harvey et al 2002;Hanson et al 2003). In a previous study, we have shown that only 1 of 15 different tetracycline-binding aptamers is able to mediate ligand-dependent regulation of gene expression .…”

Functional Aptamers and Aptazymes in Biotechnology, Diagnostics, and Therapy

“…489 Translational control elements based on tetracycline and theophylline-controlled aptamer have been realized in yeast. [490][491][492] In these studies, the potential of aptamer/ligand interactions to suppress translation was demonstrated using reporter genes ( Figure 21). …”

Control of mammalian translation by mRNA structure near caps

“…In addition, we tested GC content effects by designing fourh airpins that maintained ap redicted thermal stability near ÿ 30 kcal/mol and a+ 4 position. Insert lengths were maintained using CAA motifs as spacers, since these motifs are presumed to have no secondary structure (Hanson et al 2003). We finally cloned control constructs composed entirely of CAA motifs for normalization to ''structure-lessR NAs''.…”