Poster abstracts
Poster number 108 submitted by Dr. Nizar Saad
Understanding the regulatory mechanisms of asparagine and aspartate T box riboswitches in Clostridium acetobutylicum
Nizar Y. Saad (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Bettina Schiel (Institute of Microbiology and Biotechnology, Ulm University, D-89069 Ulm. Albert Einstein-Allee 11), Peter Drre (Institute of Microbiology and Biotechnology, Ulm University, D-89069 Ulm. Albert Einstein-Allee 11), Hubert D. Becker (Unite Mixte de Recherche 7156 Genetique Moleculaire Genomique Microbiologie, Centre National de la Recherche Scientifique, Universite de Strasbourg, F-67084 Strasbourg, France), Tina M. Henkin (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA)
Abstract:
The T box riboswitch functions primarily at the level of transcription attenuation and controls the expression of downstream amino acid-related genes by sensing the aminoacylation state of a cognate tRNA [1]. When cells are limited for the cognate amino acid, the tRNA is uncharged and binds to the T box riboswitch, which promotes formation of an antiterminator structure that allows transcription of the downstream gene.
The C. acetobutylicum genome contains two aspartyl-tRNA synthetase genes (aspS2o and aspS2), encoding DRS2o and DRS2, respectively. We previously showed that DRS2o charges tRNAAsp (aspartate) and tRNAAsn (asparagine) with Asp [2, 3]. The mischarging of Asp onto tRNAAsn is followed by Asn-tRNAAsn synthesis through the amidation of Asp by the transamidation pathway [4]. C. acetobutylicum lacks active Asn synthetase, and is unable to synthesize Asn from Asp independent of tRNAAsn [3]. We previously showed that a T box riboswitch regulates the expression of the aspS2o operon, which encodes DRS2o and the amidotransferase complex, forming a functional transamidation pathway [3]. This T box riboswitch senses the tRNAAsn aminoacylation state (NT box). C. acetobutylicum possesses an alternative route for Asn-tRNAAsn synthesis through an AsnRS that directly charges Asn onto tRNAAsn. We recently demonstrated that DRS2 is also capable of charging Asp onto tRNAAsn. Asn and Asp may both affect NT box antitermination, since their availability may affect the aminoacylation state of tRNAAsn. We previously showed that Asn affects the expression of the aspS2o operon through the NT box riboswitch [3], but the effect of Asp availability has not been investigated. We recently observed that expression of the aspS2o operon is up-regulated and that of aspS2 gene is down-regulated in C. acetobutylicum grown in minimal medium containing Asp, relative to their expression during growth in minimal medium lacking all amino acids. The expression of aspS2 is regulated by a T box riboswitch that responds to tRNAAsp (DT box). Transcriptome analysis showed that the aspS2o operon is induced in an aspS2 knock-out strain grown in minimal medium lacking all amino acids. These results suggest that the expression of aspS2 and aspS2o operon may be related. In this study, we investigate in detail the nature of this relation.
References:
1. Henkin, T. M. (2014). The T box riboswitch: A novel regulatory RNA that utilizes tRNA as its ligand. Biochim Biophys Acta.
2. Charron, C., Roy, H., Blaise, M., Giege, R., and Kern, D. (2003). Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain. Embo J 22, 1632-1643.
3. Saad, N. Y., Schiel, B., Braye, M., Heap, J. T., Minton, N. P., Durre, P., and Becker, H. D. (2012). Riboswitch (T-box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis. J Biol Chem 287, 20382-20394.
4. Curnow, A. W., Hong, K., Yuan, R., Kim, S., Martins, O., Winkler, W., Henkin, T. M., and Soll, D. (1997). Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc Natl Acad Sci U S A 94, 11819-11826.
Keywords: T box riboswitch, amino acids, aminoacyl-tRNA synthetases