Molecular analysis of a synthetic tetracycline-binding riboswitch.

Riboswitches are newly discovered regulatory elements that consist solely of RNA, sense their ligand in a preformed binding pocket, and perform a conformational switch in response to ligand binding, resulting in altered gene expression. Regulation by a tetracycline (tc)-binding aptamer when inserted into the 5' untranslated region (UTR) of a reporter gene exhibits all characteristics of a riboswitch. Chemical and enzymatic probing reveals that the aptamer consists of two stems, P1 and P2, which are already present in the absence of tc and form the scaffold of the aptamer. They are separated by a bulge B1-2 and an opposing stem-loop (P3-L3). Tc-dependent changes in the probing pattern only appear in the upper part of the bulge B1-2 (nucleotides 9-13) and the loop L3. Saturating mutagenesis corroborates the involvement of these two regions in regulation. Structural probing of the mutant A55U, which contains a single-nucleotide exchange in loop L3 results in a changed probing pattern of the loop, but also of the opposing bulge B1-2. This denotes that both regions cooperate and form a composite binding pocket. Thus, our model for aptamer-mediated translational regulation is that the ligand-free aptamer has only marginal influence on translational initiation. Tc then leads to an intramolecular connection in a pseudoknot-like manner and turns the aptamer into its inhibitory form. This represents a new mechanism for riboswitch action clearly distinguished from currently known naturally occurring riboswitches, which function by sequestration of the ribosomal binding site, transcriptional attenuation, and ribozyme-mediated degradation.

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