Talk abstracts

Talk on Friday 03:00-03:15pm submitted by Alex Runyon

Probing the structure and function of the Bacillus subtilis thrS T-box riboswitch

Alexander T. Runyon (Microbiology Department, The Ohio State University), Tina M. Henkin (Microbiology Department, The Ohio State University)

Abstract:
Many amino acid-related genes in Gram-positive bacteria are regulated at the level of transcription attenuation by cis-acting RNA structures known as T-box riboswitches. A specific uncharged tRNA that corresponds to the amino acid specificity of the downstream gene is used as the regulatory ligand. ~95% of T-box riboswitches are predicted to fold into the canonical pattern, which includes a set of conserved sequence and structural elements. Current biochemical understanding of T-box riboswitch function derives from noncanonical models that lack one or more of these 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 comparisons with biochemical data obtained from non-canonical riboswitches. Point mutations have been generated in conserved elements in thrS and in vitro transcription, tRNA binding assays, and RT-qPCR indicate that the conserved elements in thrS are critical for tRNA-dependent antitermination activity, a subset of which are critical for tRNA binding. Structural studies performed with SHAPE confirm the overall structural pattern and mutational disruption of conserved elements. 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 provide 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 USA 115:3894–3899.
Suddala KC, Zhang J. 2019. High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nat Struct Mol Biol 26:1114–1122.
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