In vitro structure-function studies of the Bacillus subtilis tyrS mRNA antiterminator: evidence for factor-independent tRNA acceptor stem binding specificity

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Abstract
Expression of many aminoacyl-tRNA synthetase, amino acid biosynthesis and transport genes in Bacillus subtilis is controlled at the level of transcription termination using the T box system and requires the formation of specific secondary structures in the mRNA leader region. One structure functions as a transcriptional terminator, while an alternate form, the antiterminator, is necessary for transcription of the downstream coding regions. We have investigated the interaction of antiterminator model RNAs, based on the B.subtilis tyrS antiterminator with tRNATyr and tRNA acceptor stem models, using a gel shift assay. Binding of the antiterminator RNA to tRNATyr was dependent on complimentarity with the acceptor end of the tRNA or microhelix; affinity for the microhelix RNA was reduced relative to the tRNA. Alteration of a conserved position in the non-base pairing region of the bulge greatly reduced tRNA binding, consistent with in vivo studies. Therefore, it appears that some of the antiterminator–tRNA binding specificity is dependent on the structure of the antiterminator bulge alone and the complex it forms with tRNA in the absence of additional trans-acting factors. During the course of these studies we also discovered that the antiterminator can form a ‘kissing’ bulge complex, a unique RNA motif. The ease of formation of this RNA homodimer illustrates the propensity for the bulge of the antiterminator to bind RNA.