RNA: Structure, Biophysics and Physiology

National Heart, Lung, And Blood Institute

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Abstract

In the past year, we made an important advance in understanding the mechanism of action of a widespread riboswitch class; this finding also has broad implications in how a large class of non-coding RNAs can regulate protein synthesis. The yjdF class of bacterial riboswitches is unique among this type of cis-regulatory mRNA elements because rather than recognizing one specific small molecule with high affinity and selectively, it binds to a broad class of small molecules (azaaromatic compounds) and it selectively activates translation only in response to a subset of its ligands. Thus, it is an RNA analog of protein transcription factors that can respond to multiple small molecules (multi-drug resistance protens). Because of this unique ability, and because it is an obvious target for the development of novel antibiotics, the yjdF riboswitch has been the target for numerous structure-determination efforts; thus far, no high-resolution structures have been forthcoming. We subjected the yjdF riboswitch to small-angle X-ray scattering, and discovered that in the presence of its activating small molecules, it adopts an overall L-shape, similar (at the resolution of the technique) to the canonical fold of tRNA. When we re-examined the secondary structure of the yjdF riboswitch in this light, it became apparent that its sequence is highly similar to that of tRNA-Lys. To further examine this putative similarity to tRNA, we engineered the yjdF riboswitch by extracting its aptamer domain from its mRNA context, and appending a CCA 3'-terminus. Remarkably, this chimeric RNA supports protein synthesis in vitro when aminoacylated and bound to ligands that activate the yjdF riboswitch (but not non-activating ligands). Consistent with this, filter binding assays demonstrated that the activator-bound yjdF riboswitch (but not the unliganded form) specifically binds to 70S ribosomal particles in filter binding assays. To examine whether this binding can directly activate translation in addition to the ligand-dependent uncovering of the Shine-Dalgarno sequence that the yjdF riboswitch is known to employ, we generated chimeric mRNAs in which the yjdF riboswitch aptamer domain was replaced by various authentic tRNA sequences. This experiment revealed that some tRNA sequences can strongly activate translation in this cis-acting context, possibly by association with the ribosomal E-site. Because tRNA-like domains and tRNA mimics are widespread among bacterial, eukaryal and viaral non-coding RNAs, the ribosome-mediated mechanism of action of the yjdF riboswitch we have uncovered may be widespread in the transcriptome, beyond suggesting a new mechanism of action for bona fide tRNAs.