Many genes involved in amino acid metabolism in Bacillus subtilis and related organisms are regulated by the T box mechanism, a novel transcription termination control system. Expression of each gene in this family depends on binding of a specific uncharged tRNA to the nascent RNA transcript, resulting in readthrough of a termination signal that otherwise attenuates downstream gene expression. tRNA recognition requires pairing of the tRNA anticodon with a single codon, the """"""""Specifier Sequence,"""""""" in the leader RNA, as well as pairing of the acceptor end of the tRNA to an antiterminator element that competes with formation of the terminator helix;these pairing are necessary but not sufficient for antitermination. tRNAGly-dependent antitermination of the B. subtilis glyQS gene can be reproduced in a purified system, and binding of tRNAGly to glyQS leader RNA results in structural changes throughout the RNA. The ability of T box RNAs to directly and specifically recognize their cognate effector molecule to regulate gene expression places these RNAs in the riboswitch family of regulatory RNAs. The next project period will be directed toward detailed analysis of the molecular mechanism of tRNA-dependent antitermination, and specific tRNA recognition by T box RNAs. We will focus on RNA elements distinct from the known regions of base-pairing, and functional differences between the glyQS model RNA, which represents a natural deletion variant, and the more common complex T box RNAs that contain additional structural elements, the function of which is unknown. We will also investigate the mechanistic difference between T box elements that operate at the level of transcription termination (found primarily in Firmicutes), and those that operate at the level of translation initiation (found primarily in Actinomycetes, including Mycobacterium sp.). Further characterization of the T box mechanism is essential to the long-term goal of developing novel antimicrobial agents that target this mechanism, which is found in many Gram-positive pathogens. The role of RNA as a regulatory molecule has been increasingly appreciated in recent years, and the T box system represents a unique molecular mechanism for RNA-mediated gene regulation, and a new function for tRNA in the cell.

Public Health Relevance

RNA-mediated regulation has recently emerged as a central player in all organisms. This study is directed toward analysis of the T box system, a unique regulatory mechanism in which RNA transcripts directly sense a specific tRNA molecule to control gene expression. The T box mechanism is widely used in Gram-positive bacteria, including important pathogens, to regulate multiple essential genes, and a long-term goal of this work is the identification of novel antimicrobial agents that inactivate this system, therefore preventing growth.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Bender, Michael T
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Ohio State University
Schools of Arts and Sciences
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Sherwood, Anna V; Frandsen, Jane K; Grundy, Frank J et al. (2018) New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch. Proc Natl Acad Sci U S A 115:3894-3899
Kreuzer, Kiel D; Henkin, Tina M (2018) The T-Box Riboswitch: tRNA as an Effector to Modulate Gene Regulation. Microbiol Spectr 6:
Sherwood, Anna V; Henkin, Tina M (2016) Riboswitch-Mediated Gene Regulation: Novel RNA Architectures Dictate Gene Expression Responses. Annu Rev Microbiol 70:361-74
Caserta, Enrico; Liu, Liang-Chun; Grundy, Frank J et al. (2015) Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME. J Biol Chem 290:23336-47
Liu, Liang-Chun; Grundy, Frank J; Henkin, Tina M (2015) Non-Conserved Residues in Clostridium acetobutylicum tRNA(Ala) Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch. Life (Basel) 5:1567-82
Sherwood, Anna V; Grundy, Frank J; Henkin, Tina M (2015) T box riboswitches in Actinobacteria: translational regulation via novel tRNA interactions. Proc Natl Acad Sci U S A 112:1113-8
Williams-Wagner, Rebecca N; Grundy, Frank J; Raina, Medha et al. (2015) The Bacillus subtilis tyrZ gene encodes a highly selective tyrosyl-tRNA synthetase and is regulated by a MarR regulator and T box riboswitch. J Bacteriol 197:1624-31
Henkin, Tina M (2014) The T box riboswitch: A novel regulatory RNA that utilizes tRNA as its ligand. Biochim Biophys Acta 1839:959-963
Grigg, Jason C; Chen, Yujie; Grundy, Frank J et al. (2013) T box RNA decodes both the information content and geometry of tRNA to affect gene expression. Proc Natl Acad Sci U S A 110:7240-5
Caserta, Enrico; Haemig, Heather A H; Manias, Dawn A et al. (2012) In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor protein. J Bacteriol 194:3386-94

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