Poster number 122 submitted by Alex Runyon
Understanding canonical T-box riboswitches: A study of the Bacillus subtilis thrS RNA
Alexander T. Runyon (Microbiology Department, The Ohio State University), Jane K. Frandsen (Ohio State Biochemistry Program, The Ohio State University), Rebecca N. Wiliams-Wagner (Microbiology Department, The Ohio State University), Tina M. Henkin (Microbiology Department, The Ohio State University)
T-box riboswitches regulate many amino acid-related genes in Gram-positive bacteria. These cis-acting RNA structures utilize an uncharged tRNA corresponding to the downstream gene as a regulatory ligand. Roughly 95% of T-box RNAs are predicted to adopt a canonical structure, composed of several RNA motifs. Previous biochemical and structural studies of T-box riboswitches have focused primarily on T-box RNAs that lack one or more conserved structural elements, as in vitro work with a canonical T-box RNA has not been possible. We have demonstrated specific tRNAThr binding to the canonical Bacillus subtilis thrS T-box RNA and tRNAThr-dependent antitermination in a purified in vitro system. Point mutations were generated in conserved RNA motifs of the thrS riboswitch, and the structural and functional effects were investigated using SHAPE, in vitro transcription assays, tRNA binding assays, and in vivo RT-qPCR assays. These studies revealed that all conserved elements, including those absent from the noncanonical T-box RNAs, are required for function both in vivo and in vitro. Our analysis of the thrS riboswitch has provided a more robust understanding of how all of the key structural elements interact in canonical T-box RNAs, which represent the majority of these RNAs found in nature.
1 Grundy FJ, Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482.
2 Sherwood AV, Frandsen JK, Grundy FJ, Henkin TM. 2018. New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch. Proc Natl Acad Sci USA 115:3894–3899.
3 Putzer H, Condon C, Brechemier-Baey B, Brito R, Grunberf-Manago M. 2002. Transfer RNA‐mediated antitermination in vitro. Nucleic Acids Res. 30:3026–3033.
4 Suddala KC, Zhang J. 2019. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122.
5 Rollins SM, Grundy FJ, Henkin TM. 1997. Analysis of cis-acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene. Mol Microbiol 25:411–421.
Keywords: tRNA, Riboswitch, T-box