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

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Hellmann, Rachel Alice

Contributors dc:contributor

• Martinis, Susan A.

• Chemistry, Biochemistry

Identifier

(UMI)AAI3362919

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/72340