Poster abstracts

Poster number 16 submitted by Janie Frandsen

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch

Jane K. Frandsen (Ohio State Biochemistry Program, Cellular, Molecular & Biochemical Sciences Program, Center for RNA Biology), Anna V. Sherwood (Molecular Cellular and Developmental Biology, Center for RNA Biology), Frank J. Grundy (Department of Microbiology, Center for RNA Biology), Tina M. Henkin (Department of Microbiology, Center for RNA Biology)

Abstract:
T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes.1 Expression of T box family genes is downregulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA.2,3 Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs, and the roles of several conserved structural domains are still unknown.3,4 In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNAIle ligand. Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) showed structural changes in the conserved riboswitch domains upon interaction with the ligand tRNA. Cross-linking and mutational analyses identified two new interaction sites, one between the S-turn element in Stem II and the T arm of tRNAIle and the other between the Stem IIA/B pseudoknot and the D loop of tRNAIle. These newly identified RNA contacts add new features of tRNA recognition by the T box riboswitch and demonstrate a new role for the S-turn and pseudoknot elements that are common to many cellular RNAs.

References:
1. Gutiérrez-Preciado A, Henkin TM, Grundy FJ, Yanofsky C, Merino E. Microbiol Mol Biol Rev 73:36–61 (2009).
2. Grundy FJ, & Henkin TM. Cell 74: 475-82 (1993).
3. Sherwood AV, Grundy FJ, Henkin TM. Proc. Natl. Acad. Sci. USA 112: 1113-18 (2015).
4. Henkin TM. Biochim. Biophys. Acta 1839: 959-963 (2014).

Keywords: Riboswitch, tRNA, S-turn, Pseudoknot, T arm, D loop