Poster abstracts
Poster number 49 submitted by Janie Frandsen
Non-canonical T-box riboswitch-tRNA recognition in ileS variants
Jane K. Frandsen (Ohio State Biochemistry Program, Center for RNA Biology), Anna V. Sherwood (Molecular Cellular and Developmental Biology, Center for RNA Biology), Alexandar L. Hansen (CCIC NMR Facility), Frank J. Grundy (Department of Microbiology, Center for RNA Biology), Mark P. Foster (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for RNA Biology), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology, Center for RNA Biology)
Abstract:
T-box riboswitches regulate the expression of amino acid-related genes by monitoring the ratio of charged to uncharged cognate tRNA. The ileS T-box riboswitches, which regulate isoleucyl-tRNA synthetase genes, have three classes of Stem I domains: canonical Stem I, Ultrashort Stem I (US), and Unusually Structured Stem I Region (USSR).1 In canonical T-box RNAs, the Specifier Loop interacts with the anticodon of the cognate tRNA and the Stem I terminal loop and AG bulge form a loop-loop structure that interacts with the tRNA elbow.2,3 Many medically relevant bacteria, including Mycobacterium sp., have a US-type RNA that lacks elements necessary for the interaction with the tRNA elbow. We hypothesize that in the absence of a canonical Stem I, other structural elements of the riboswitch facilitate interactions between the leader RNA and tRNA. In this study, we investigate the structural changes in US Stems I and II induced by tRNAIle binding. Fluorescence quenching experiments with 2-aminopurine substituted RNA suggest that cognate tRNA binding induces comparable base stacking in the Specifier Loop of both canonical and US Stem I classes. Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) experiments indicate that cognate tRNA binding stabilizes the k-turn structure at the base of Stem I and induces structural changes in the Stem II S-turn. Homonuclear and heteronuclear 2D NMR experiments show that the k-turn affects the structure of the Specifier Loop and that tRNA addition induces structural rearrangements in the Specifier Loop. Together, these results indicate that the k-turn and S-turn play important roles in tRNA binding to the US T-box riboswitch.
References:
1. Sherwood AV, Grundy FJ, Henkin TM. Proc. Natl. Acad. Sci. USA 112: 1113-18 (2015).
2. Grundy FJ, & Henkin TM. Cell 74: 475-82 (1993).
3. Grigg JC, Chen Y, Grundy FJ, Henkin TM, Pollack L, Ke A. Proc. Natl. Acad. Sci. USA 110: 7240-45 (2013).
Keywords: T-box riboswitch, NMR, SHAPE