Amino acid editing and orthogonal enzyme complementation screen by class II prolyl-tRNA synthetase.

摘要

My thesis research is primarily focused on amino acid editing by class II Saccharomyces cerevisiae prolyl-tRNA synthetase (ProRS) and orthologous enzyme complementation in vivo. Aminoacyl-tRNA synthetases generally contain an ancient catalytic core domain and one or more "extra" domains that function to enhance the specificity of genetic code translation. Through sequence alignment of the Haemophilus influenzae YbaK protein, it had been demonstrated that yeast N-terminal share strong structural similarities with the Escherichia coli INS domain. Using biochemistry and a multitude of molecular biology techniques, I have shown that yeast ProRS possesses pre-transfer editing activity and lacks the ability to hydrolyze mischarged Ala-tRNAPro species. We have also demonstrated that the pre-transfer editing activities of ProRS are a functional part of the synthetic active site. Though Sc ProRS does not appear to possess post-transfer editing activity in vitro, there are strong structural similarities between the Ec INS domain and the Sc N-terminus, suggesting that the latter may be the remnant of a defunct editing domain. Furthermore, I have shown that yeast ProRS, when fused to the E. coli INS domain in cis, the yeast ProRS Chimera is now able to hydrolyze Ala-tRNAPro, demonstrating an unusual gain of posttransfer editing function. Importantly, this work has raised concerns about conditions that may necessitate editing as well as consensus sequences that may predict ProRS editing activity.