REGULATION OF BACILLUS SUBTILIS TRNA SYNTHETASE GENES
Principal Investigator: TINA HENKIN
Affiliation: The Ohio State University
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: -------------------- - --------------------
more information: NIH RePORT
- An intergenic stem-loop mutation in the Bacillus subtilis ccpA-motPS operon increases motPS transcription and the MotPS contribution to motilityNaoya Terahara
Graduate School of Life Sciences, Toyo University, 1 1 1 Izumino, Itakura machi, Oura gun, Gunma 374 0193, Japan
J Bacteriol 188:2701-5. 2006....
- T box RNA decodes both the information content and geometry of tRNA to affect gene expressionJason C Grigg
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA
Proc Natl Acad Sci U S A 110:7240-5. 2013..When both conditions are met, tRNA is secured, and its aminoacylation state is sensed...
- Riboswitch RNAs: using RNA to sense cellular metabolismTina M Henkin
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA
Genes Dev 22:3383-90. 2008..Regulation occurs as a consequence of direct binding of an effector molecule, or through sensing of a physical parameter such as temperature. Here we review the global role of riboswitch RNAs in bacterial cell metabolism...
- 4,5-Disubstituted oxazolidinones: High affinity molecular effectors of RNA functionRajaneesh Anupam
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
Bioorg Med Chem Lett 18:3541-4. 2008..Novel 4,5-disubstituted oxazolidinones were identified as high affinity RNA molecular effectors that modulate the transcription antitermination function of the T box riboswitch...
- Control of acetyl-coenzyme A synthetase (AcsA) activity by acetylation/deacetylation without NAD(+) involvement in Bacillus subtilisJeffrey G Gardner
Department of Bacteriology, 144A Enzyme Institute, 1710 University Avenue, Madison, WI 53726 4087, USA
J Bacteriol 188:5460-8. 2006..Unlike sirtuins, the AcuC protein did not require NAD(+) as cosubstrate to deacetylate AcsA(Ac). The function of the putative AcuB protein remains unknown...
- tRNA regulation of gene expression: interactions of an mRNA 5'-UTR with a regulatory tRNAAudrey R Nelson
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695 7622, USA
RNA 12:1254-61. 2006..2 +/- 1.4 microM). Thus, the bimolecular 5'-UTR and ASL(Gly)(GCC) models mimic the RNA-RNA interaction required for T box gene regulation in vivo...
- In vitro approaches to analysis of transcription terminationIrina Artsimovitch
Department of Microbiology and Center for RNA Biology, The Ohio State University, 484 W 12th Avenue, Columbus, OH 43210, USA
Methods 47:37-43. 2009..We describe a basic protocol for analysis of transcription termination in vitro, and include descriptions of parameters that can be modified for specific types of experimental questions...
- In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor proteinEnrico Caserta
Department of Microbiology, The Ohio State University, Columbus, Ohio, USA
J Bacteriol 194:3386-94. 2012..The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo...
- NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNAJiachen Wang
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251 1892, USA
Nucleic Acids Res 38:3388-98. 2010....
- Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanismChristopher M Johnson
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA
J Bacteriol 192:1634-42. 2010..Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication)...
- Riboswitch RNAs: regulation of gene expression by direct monitoring of a physiological signalAngela M Smith
Department of Microbiology and Center for RNA Biology, Ohio State University, Columbus, OH, USA
RNA Biol 7:104-10. 2010..An overview of the role of riboswitch RNAs in bacterial gene expression will be provided, and a few examples are described in more detail to illustrate the types of mechanisms that have been uncovered...
- The T box mechanism: tRNA as a regulatory moleculeNicholas J Green
Department of Microbiology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
FEBS Lett 584:318-24. 2010..Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA...
- Analysis of tRNA-directed transcription antitermination in the T box system in vivoTina M Henkin
Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA
Methods Mol Biol 540:281-90. 2009....
- Biochemical features and functional implications of the RNA-based T-box regulatory mechanismAna Gutiérrez-Preciado
Department of Molecular Microbiology, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos 62210, Mexico
Microbiol Mol Biol Rev 73:36-61. 2009....
- RNA-dependent RNA switches in bacteriaTina M Henkin
Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA
Methods Mol Biol 540:207-14. 2009..This review will summarize these classes of regulatory mechanisms and their use in bacterial systems...