Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases (aaRS), is essential to insure translational fidelity. Here, it is shown that expression of AspRS is regulated in Saccharomyces cerevisiae by a feedback mechanism, that necessitates the binding of AspRS to its messenger RNA. The correlation between AspRS expression and mRNAAspRS and tRNAAsp concentrations, as well as the presence of AspRS in the nucleus, suggest an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNAAsp, is imported in the nucleus where it binds to mRNAAspRS and thus inhibits its accumulation. <br />We have established the folding of the 300-nucleotides long 5' end of mRNAApRS and identified the structural signals involved in the regulation process. We propose that the mRNAAspRS fragment folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNAAsp anticodon-like stem-loop structure that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, support a model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure like it recognizes its cognate tRNAAsp.<br />Finally, the consequences of an increased concentration of AspRS in the cell have been tested. In vitro, high AspRS concentrations lead to mis-aspartylation of tRNAAsn and tRNAGlu. In vivo, the design of a reporter gene conferring an antibiotic resistance, dependent on mischarged tRNAs, did not allow to detect any cross aminoacylation. However, the proteomic analysis of yeasts overexpressing AspRS pointed out the conditions of AspRS accumulation in the cell by detecting the presence of an additional control mechanism at the post-translational level.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-00139129 |
| Date | 13 October 2006 |
| Creators | Ryckelynck, Michael |
| Publisher | Université Louis Pasteur - Strasbourg I |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | French |
| Detected Language | English |
| Type | PhD thesis |
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