Date of Graduation
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Second Committee Member
Third Committee Member
Aminoacyl-tRNA synthetases (AARSs), Enzymes, Genetic code expansion, Lysine acetylation, Mistranslation
Aminoacyl-tRNA synthetases (AARSs) are an ancient and highly conserved family of enzymes which can catalyze a two-steps aminoacylation reaction to charge tRNAs with their cognate amino acids, thus playing crucial roles in ribosomal protein synthesis. Naturally, the accurate amino acids and tRNA recognition of these synthetases are essential to the fidelity of translation process. To assure the correct recognition, some of these synthetases have evolved with an editing function to help remove the mischarged tRNAs. In addition to these functions, AARSs are also involved in various biological processes ranging from transcription to translation. Currently, a series of proteomic studies have shown that AARSs are one of the enzymes with the most abundant acetylation. And a few studies have shown site-specific acetylation on AARSs can cause significant changes in enzyme activities. It is necessary to conduct more investigation on the relationship between acetylation and the function of AARSs.
However, due to the dynamic, reversible, transient, and uncontrollable pattern of lysine acetylation, it is usually challenging to learn its effects on proteins. To solve this problem, genetic code expansion strategy can be an ideal solution. This strategy offers a co-translational incorporation system (a pair of engineered orthogonal AARS/tRNA and an unassigned codon is required) which is able to introduce noncanonical amino acids including acetyllysine (AcK) into specific positions of object proteins.
Using an engineered pyrrolysyl-tRNA synthetase variant specific for AcK and its orthogonal tRNApyl, this study established an AcK incorporation system which could stably produce site-specific acetylated AARSs. Four AARSs in Escherichia coli, including class I cysteinyl-tRNA synthetase (CysRS) as well as class II aspartyl-tRNA synthetase (AspRS), histidyl-tRNA synthetase (HisRS), and threonyl-tRNA synthetase (ThrRS), were chosen as objects in this study. The results indicate that the impacts of lysine acetylation could be different between two classes, and even within the class. In addition, this study also found that acetylation of ThrRS (at K169) could affect its editing function can cause mistranslation. To furtherly detect the possible mistranslation rate of ThrRS with site-specific acetylation in its editing domain, we also designed a GFP variant with only one Threonine (Thr) in position 203 which determines the fluorescence signal of GFP. Briefly, if the mistranslation occurs, then the Thr would be mistranslated to serine, and then the GFP probe would lose its fluorescence signal. This probe was then applied to indicate that K169 acetylation could affect the editing function of ThrRS and cause mistranslation in vivo.
Chen, H. (2021). Site-specific Effects of Lysine Acetylation on Aminoacyl-tRNA Synthetase. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4356