The T box transcription antitermination system is a riboswitch found primarily in Gram-positive bacteria which monitors the aminoacylation of the cognate tRNA and regulates a variety of amino acid-related genes. Novel 4,5-disubstituted oxazolidinones were identified as high affinity RNA molecular effectors that modulate the transcription antitermination function of the T box riboswitch.Identifying RNA ligands that modulate transcription regulation is an important area for drug discovery that has been only minimally explored to date. One potential therapeutic target is the T box transcription antitermination mechanism. This mechanism regulates many amino acid-related genes, including aminoacyl-tRNA synthetase genes, and is found predominantly in Gram-positive bacteria. 1 The T box RNAs are members of the "riboswitch" family in which nascent RNAs directly sense effector molecules to control gene expression. 2-4 The T box genes contain a complex set of structural elements within the 5′ untranslated region of their mRNAs (the "leader region"). These elements include a transcription termination signal that abrogates synthesis of the full-length mRNA and a competing antiterminator element. Readthrough of the terminator, and expression of the downstream gene, is dependent on binding of a specific uncharged tRNA to the nascent RNA transcript; each gene in the T box family responds independently to the cognate uncharged tRNA. 5 The T box antitermination mechanism can function in the absence of additional cellular factors, 6 and the antiterminator RNA element is a critical component of the mechanism. 5 The leader RNA-tRNA interaction stabilizes the antiterminator element, thereby preventing formation of the competing terminator element (Figure 1). The antiterminator element is highly conserved and has been extensively characterized by genetic, biochemical and structural biology approaches. 7-9 A significant challenge in rational ligand design for RNA structure-specific binding is to achieve both high affinity and excellent tertiary structure specificity. Aminoglycosides, the most widely studied RNA ligands, bind primarily in divalent cation binding sites. 10-12 The electrostatic attraction between the multiple protonated amino groups and the negatively charged RNA phosphate backbone leads to very high affinities. However, due to the ubiquitous presence of divalent cation binding sites in RNA, primarily for tertiary fold stabilization, 13 the aminoglycosides readily bind many RNAs 14 thus reducing their utility for RNA structurespecific ligand design. A variety of other RNA ligands have been investigated, 15-21 but few Correspondence to: Jennifer V. Hines. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the produ...

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The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized and their binding to the T-box riboswitch antiterminator model RNA investigated in detail. Characterization of ligand affinities and binding site localization indicate that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets.

A unique RNA-RNA interaction occurs between uncharged tRNA and the untranslated mRNA leader region of bacterial T box genes. The interaction results in activation of a transcriptional antitermination molecular switch (riboswitch) by stabilizing an antiterminator RNA element and precluding formation of a competing transcriptional terminator RNA element. The stabilization requires the base pairing of cognate tRNA acceptor end nucleotides with the antiterminator. To develop an appropriate model system for detailed structural studies and to screen for small molecule disruption of this important RNA-RNA interaction, steady-state fluorescence measurements of antiterminator model RNAs were used to determine the dissociation constant for model tRNA binding. The antiterminator-binding affinity for the full, minihelix, microhelix, and tetramer tRNA models differed by orders of magnitude. In addition, not all of the tRNA models exhibited functionally relevant binding specificity. The results from these experiments highlight the importance of looking beyond the level of known base pairing interactions when designing functionally relevant models of riboswitch systems.