Origins and Evolution of tRNA Synthetase Fidelity Modules

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

Intellectual Merit: The tRNA synthetases (aaRSs) are enzymes responsible for the first and most crucial step of protein synthesis, covalent attachment of the correct amino acid to its cognate tRNA. The tRNA then delivers the attached amino acid to the ribosome for insertion into the correct place in each protein that is synthesized, guided by the mRNA template. Accurate protein synthesis is essential to the survival of all living organisms and requires that aaRS enzymes attach the correct amino acid with high accuracy. For about half of the amino acids, the corresponding aaRS enzyme has difficulty selecting the correct amino acid because at least one other amino acid is similar in size and chemical nature and occasionally is incorporated in place of the correct one. These aaRS enzymes have evolved proofreading or editing modules to correct errors in amino acid selection. Mistakes in protein synthesis can be very harmful to cellular function and may even result in cell death. This project focuses on two aaRS enzymes that have editing modules. Leucyl-tRNA synthetase (LeuRS) has an editing module called CP1. Computation and biochemistry were used to determine that LeuRSs from three different Mycoplasma species contain mutations in the CP1 domain that compromise its editing activities. Phenylalanyl- and threonyl-tRNA synthetases from Mycoplasma also appear to lack functional editing modules. These aaRS editing-defects translated into errors in protein synthesis. This project will test the hypothesis that the loss of editing function in these enzymes is actually beneficial to these organisms and have been selected by evolution because proteins with random mutations provide a benefit to these parasitic organisms. The benefit of statistical errors in protein sequences will be probed in this project using more facile non-parasitic model bacteria. Sulfolobus are bacteria that thrive in hot springs that are subject to fluctuating high temperatures and acidic environmental conditions. All known Sulfolobus species contain two, apparently fully functional, copies of the LeuRS gene. What distinguishes these two genes is that one contains modifications in the editing module, expected to disrupt its error correction mechanisms. It is likely that this secondary LeuRS lacks fidelity. Experiments with Sulfolobus will be carried out to investigate if decreased aaRS fidelity is maintained as a selective advantage. Yeast mitochondrial LeuRS has an adapted CP1 editing domain that is responsible for an essential RNA splicing activity. Co-evolution of the CP1 domain for RNA splicing occurred at a cost to its fidelity function and is consistent with published experiments showing that yeast mitochondria can tolerate LeuRS editing defects. The mechanistic and molecular co-adaptation of LeuRS and its CP1 editing domain for splicing will be investigated.It has been shown that naturally occurring protein synthesis errors are produced in organisms and that they can be dependent on error-prone aaRSs. This was paradigm-shifting because it demonstrated that mutations that are 'statistical' can be tolerated and perhaps even preferred by certain organisms. The intellectual merit of this project is a focus on new models to investigate cellular benefits that occur when aaRS editing is compromised, resulting in decreased thresholds of fidelity and statistical errors. It will also investigate the evolutionary and functional co-adaptation of an aaRS editing domain for secondary functions in the cell. Broader Impacts: It was shown that unusual aaRSs produce mischarged tRNAs for the purpose of generating proteins with statistical errors. This broadly impacts the field of evolution and current beliefs of cellular dependence on translational fidelity. This project will develop models to probe the benefits and selective advantage of decreased fidelity for the cell. A portion of this project is dedicated to outreach efforts to young children in their school classrooms to improve science literacy across a broad spectrum of students. Academic modules with an experimental component will introduce students to microbiology and the three kingdoms of life. This module is designed to educate young children in fundamental biology concepts and would be expected to broadly impact and stimulate their interest in science. In addition, graduate and undergraduate training and mentoring will be integrated throughout the research and education plan to enhance their professional and scientific development as well as to provide role models to children and also junior researchers.