| Popis: |
When the sequence of amino acids in a newly synthesized protein is not the same as the genetically encoded sequence, a gene is said to have been mistranslated. Alterations in protein sequence resulting from errors in genome maintenance and expression occur with low frequency. The next step in gene expression, protein synthesis, offers the greatest opportunity for errors, with mistranslation events routinely occurring at a frequency of ~1 per 10,000 mRNA codons translated. The translation of genetic information into functional proteins is a multi-step process. Aminoacyl-tRNA synthetases (aaRSs) provide the cell with substrates for protein synthesis by correctly pairing amino acids with their cognate tRNAs. Aminoacylation occurs in a two-step reaction. First, cognate amino acid is activated within the catalytic domain to form an aminoacyl-adenylate (aa-AMP). The activated amino acid is then transferred to the 3’ OH of the terminal adenosine on the tRNA acceptor stem of its cognate tRNA, forming an aminoacyl-tRNA (aa-tRNA). Phenylalanyl-tRNA synthetase (PheRS), for example, is responsible for pairing phenylalanine (Phe) with tRNAPhe. Mispaired aa-tRNA species occasionally arise due to a lack of adequate amino acid discrimination within the PheRS active site, resulting in the synthesis of misacylated Tyr-tRNAPhe. The ability of PheRS to misacylate tRNAPhe with non-cognate amino acid makes tRNA proofreading (“editing”) mechanisms of PheRS essential to maintaining the accuracy of translation.In eukaryotes, amino acid starvation activates the protein kinase Gcn2p, which leads to changes in gene expression and protein synthesis as part of a global stress response. The signal for Gcn2p activation is deacylated tRNA, which accumulates when tRNA aminoacylation is limited either through lack of substrates or inhibition of synthesis. While the primary role of aaRS editing is to prevent misaminoacylation, here we describe conditions under which editing of non-cognate aa-tRNA is also required for proper detection of amino acid starvation by Gcn2p.In order to directly determine the identity of amino acids misacylated to specific tRNAs, we developed a tRNA pulldown method which allows determination of global aminoacylation profile for a given tRNA isoacceptor. Applying this technology, we identified that PheRS lacking quality control mechanisms allowed for accumulation of Tyr-tRNAPhe (5%) but not deacylated tRNAPhe during amino acid starvation. As with the bacterial stringent response, we found that accurate monitoring of amino acid starvation in yeast is dependent on aaRS-mediated translation quality control to ensure proper accumulation of deacylated tRNA species. Our data reveal a critical function for aaRS-editing in stress responses that is independent of their role in preventing mistranslation.With regards to mistranslation, cumulative translational error rates have been determined at the organismal level, yet the extent of codon specific error rates and the spectrum of misincorporation error from system to system remain less explored. To address this deficiency, we have developed a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. Implementation of high resolution methods to determine global baselines for codon specific misincorporation events is required to shed light on these important metrics and would ultimately constitute an essential resource for the study of translation.If we consider the full spectrum of mistranslation presented herein, it becomes clear that some organisms tolerate substantial misincorporation events and that these events have the potential to confer selective fitness advantages at the organismal level. The technical advances described in Chapter 2 provide the opportunity to substantially broaden our understanding of the role of mistranslation by allowing measurement of the rates of both misaminoacylation and mistranslation in vivo. Accurate error rate measurements will provide a means to properly establish the relevance of mistranslation in physiological contexts by delineating how the level of mistranslation correlates with a cell’s ability to adapt, survive and thrive under different conditions. |
