Poster abstracts

Poster number 41 submitted by Alex Runyon

Importance of conserved elements in the Bacillus subtilis thrS T-box riboswitch

Alexander T Runyon (Department of Microbiology; Center for RNA Biology), Tina M Henkin (Molecular, Cellular, and Developmental Biology Graduate Program; Center for RNA; Ohio State Biochemistry Program; Department of Microbiology), Frank J Grundy (Center for RNA Biology; Department of Microbiology)

Abstract:
T-box riboswitches are cis-acting RNA structures that modulate expression of downstream amino acid-related genes in Gram positive bacteria. Gene expression is induced in response to a specific uncharged tRNA that corresponds to the amino acid specificity of the downstream gene, which is generally regulated through transcription antitermination. T-box riboswitches that contain the conserved elements found in the majority of T-box genes are known as canonical T-box riboswitches. Current biochemical understanding of T-box riboswitch function derives from models that lack key conserved elements. The Bacillus subtilis thrS riboswitch is located upstream of the threonyl-tRNA synthetase coding sequence and contains all of the major conserved elements, making it a canonical riboswitch. Unlike other canonical T-box RNAs, tRNA-dependent antitermination in vitro has been demonstrated with thrS, which allows for comparisons with biochemical data obtained from non-canonical riboswitches. Point mutations have been generated in conserved elements in thrS. Results from in vitro transcription and tRNA binding assays suggest that the conserved elements in thrS are critical for tRNA-dependent antitermination activity. In addition, some elements of thrS have been modified to resemble the corresponding elements of the non-canonical ileS US riboswitch found in Actinobacteria. These mutations reduce thrS function, although not as severely as point mutations that disrupt key elements. Analysis of the thrS system will allow us to gain a better understanding of the function of canonical T-box RNAs, which represent the major class of these elements found in nature.

References:
Kreuzer KD, Henkin TM. 2018. The T-Box riboswitch: tRNA as an effector to modulate gene regulation. Microbiol Spectrum 6(4).

Grundy FJ, Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475–482.

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 U S A 115:3894–3899.

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.

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: T-box, Riboswitch, tRNA