Biochemical Analysis of the Molecular Factors of Leucyl-Trna Synthetase That Ensure Aminoacylation Fidelity

160 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.The tRNA synthetases catalyze aminoacylation of transfer RNA (tRNA) with specific amino acids. Accurate aminoacylation is critical to the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS) can misactivate a broad scope of non-leucine amino acids. In these cases, mischarged tRNA products are translocated approximately 30 A from the canonical aminoacylation core of LeuRS to an editing active site within a completely separate domain called CP1. A peptide has been identified within the CP1 domain of Escherichia coli (E. coli) LeuRS, which is distal to the hydrolytic editing active site, and contributes to translocation of mischarged tRNA products. Mutational analysis of this peptide distinguished two classes of LeuRS mutants. Both classes mischarge tRNA. Surprisingly, the first class of mutants have maintained deacylation activity of mischarged tRNA as well as a robust leucylation activity. Therefore, the hypothesis is that this class of mutants charge tRNALeu, but fail to translocate these products to a competent amino acid editing site where they can be cleared. The second class of mutants are toxic to cells, which may be related to the very high in vitro mischarging activity that was measured for these mutants. Furthermore, these mutants have reduced leucylation and deacylation activity, indicating that post-transfer editing activity has been altered. It was hypothesized that this class of mutants affect the viability of aminoacyl-adenylates through an alternative editing pathway.The CP1 domain moves in relation to the aminoacylation domain as the enzyme undergoes its multi-step reaction sequence. This movement is reliant on two flexible beta-strand linkers between the two domains. The CP1 domain movement and orientation is likely facilitated by making and breaking inter-domain salt bridges between the canonical core and CP1 domain. To capture these transient interactions, cyanogen gas was employed to cross-link LeuRS peptides that could then be identified by mass spectrometry.The canonical genetic code has potentiel to be expanded by developing orthogonal aaRS:tRNA systems. Tools to generate an orthogonal LeuRSaRNA Leu pair were developed by using a two plasmid system in an E. coli strain containing a nonfunctional genomic lacZ gene with an amber stop codon. The plasmid pRAH curies tRNALeu with an amber suppressor anticodon, while pKEG harbors LeuRS. From this system, directed evolution approaches can be used to screen for an orthogonal tRNA and a LeuRS derivative that will charge it.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD