A practical guide to teaching with Proteopedia.

Proteopedia (proteopedia.org) is an open resource to explore the structure-function relationship of proteins and other biomolecules. This guide provides practical advice on how to incorporate Proteopedia into teaching the structure and function of proteins and other biomolecules. For 11 activities, we discuss desired outcomes, setting expectations, preparing students for the tasks, using resources within Proteopedia, and evaluating student work. We point out features of Proteopedia that make it especially suitable for teaching and give examples of how to avoid common pitfalls.

Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA.

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 has been investigated in detail. Characterization of ligand affinities and binding site localization indicates 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.

Fluorescence probing of T box antiterminator RNA: insights into riboswitch discernment of the tRNA discriminator base.

The T box transcription antitermination riboswitch is one of the main regulatory mechanisms utilized by Gram-positive bacteria to regulate genes that are involved in amino acid metabolism. The details of the antitermination event, including the role that Mg(2+) plays, in this riboswitch have not been completely elucidated. In these studies, details of the antitermination event were investigated utilizing 2-aminopurine to monitor structural changes of a model antiterminator RNA when it was bound to model tRNA. Based on the results of these fluorescence studies, the model tRNA binds the model antiterminator RNA via an induced-fit. This binding is enhanced by the presence of Mg(2+), facilitating the complete base pairing of the model tRNA acceptor end with the complementary bases in the model antiterminator bulge.

4,5-Disubstituted oxazolidinones: High affinity molecular effectors of RNA function.

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.

T box riboswitch antiterminator affinity modulated by tRNA structural elements.

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.

Fluorescence resonance energy transfer studies of aminoglycoside binding to a T box antiterminator RNA.

The T box transcription antitermination mechanism is found in many Gram-positive bacteria. The T box genes are typically tRNA synthetase, amino acid biosynthesis, and amino acid transport genes that have a common transcriptional control mechanism in which a unique RNA-RNA interaction occurs between an uncharged tRNA and the 5' leader region of the nascent mRNA, leading to antitermination of transcription. The tRNA binds the mRNA in at least two regions: the specifier sequence and the antiterminator. If the latter interaction does not occur, then transcription is terminated. The binding of eight different aminoglycosides to a model of the Bacillus subtilis tyrS T box antiterminator RNA has been studied using fluorescence resonance energy transfer. The observed single-site binding dissociation constants were in the low to mid micromolar range. The structure-activity relationship of aminoglycoside binding indicates that selective binding of small molecules to T box antiterminator RNA can be achieved.