The overarching objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. Accurate substrate recognition and discrimination by aminoacyl-tRNA synthetases (aaRS) is a key feature of this process. There are two classes of aaRS, and research in the Musier-Forsyth lab for the past sixteen years has revolved around class II synthetases, with a strong focus on prolyl-tRNA synthetase (ProRS). During the previous grant period, we began turning our focus to quality control mechanisms that prevent mistranslation due to the misactivation of structurally related amino acids by aaRSs. After misactivation, some noncognate amino acids are immediately hydrolyzed in the aminoacylation active site (i.e., the first or "coarse sieve") in a reaction known as "pre- transfer" editing;others are attached to tRNA and translocated to an editing site that has been designated the second or "fine sieve". The latter domain carries out a reaction referred to as "post-transfer" editing. ProRSs display an unusually diverse range of domain architectures. Bacterial enzymes contain an editing domain (INS) that is responsible for clearing misacylated Ala-tRNAPro. The INS domain is missing from most archaeal and eukaryotic enzymes, however, genome-encoded single-domain proteins (e.g., YbaK family) with homology to the ProRS INS domain are widely distributed in all three kingdoms. Interestingly, some members of this family have been shown to possess Cys-tRNAPro editing activity and to interact with the ProRS/tRNA complex. These observations allowed us to propose a novel "triple-sieve" mechanism of editing, which forms the basis of the present renewal application. Here, we propose to continue to explore the specificities and novel functions of these intriguing and poorly understood single-domain proofreading proteins, as well as their mechanisms and species-specific differences.
The specific aims are: 1) To explore pre-transfer editing by ProRS, 2) to explore the post-transfer editing mechanism of ProRS and ProRS-like editing domains, 3) to explore the function of YbaK and homologous families of proteins in vitro and in vivo, and 4) to probe the structure of the ProRS/YbaK/tRNA ternary complex.
The objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. The aminoacyl-tRNA synthetases and related genome-encoded single domain proteins studied in this work, possess editing functions that prevent errors in protein synthesis. Editing-defective synthetases have been implicated in progressive neurodegenerative diseases. Thus, in recent years, it has become clear that a better understanding of the editing functions of synthetases may provide insights and novel treatments for various diseases. Due to their essential function, these enzymes are also promising targets for the development of antibiotics and antifungal agents.
|Liu, Ziwei; Vargas-Rodriguez, Oscar; Goto, Yuki et al. (2015) Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation. Proc Natl Acad Sci U S A 112:6027-32|
|Novoa, Eva Maria; Vargas-Rodriguez, Oscar; Lange, Stefanie et al. (2015) Ancestral AlaX editing enzymes for control of genetic code fidelity are not tRNA-specific. J Biol Chem 290:10495-503|
|Dewan, Varun; Reader, John; Forsyth, Karin-Musier (2014) Role of aminoacyl-tRNA synthetases in infectious diseases and targets for therapeutic development. Top Curr Chem 344:293-329|
|Das, Mom; Vargas-Rodriguez, Oscar; Goto, Yuki et al. (2014) Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucleic Acids Res 42:3943-53|
|AlemÃ¡n, Elvin A; de Silva, Chamaree; Patrick, Eric M et al. (2014) Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle. J Phys Chem Lett 5:777-781|
|Bartholow, Thomas G; Sanford, Brianne L; Cao, Bach et al. (2014) Strictly conserved lysine of prolyl-tRNA Synthetase editing domain facilitates binding and positioning of misacylated tRNA(Pro.). Biochemistry 53:1059-68|
|Kumar, Sandeep; Das, Mom; Hadad, Christopher M et al. (2013) Aminoacyl-tRNA substrate and enzyme backbone atoms contribute to translational quality control by YbaK. J Phys Chem B 117:4521-7|
|Johnson, James M; Sanford, Brianne L; Strom, Alexander M et al. (2013) Multiple pathways promote dynamical coupling between catalytic domains in Escherichia coli prolyl-tRNA synthetase. Biochemistry 52:4399-412|
|Vargas-Rodriguez, Oscar; Musier-Forsyth, Karin (2013) Exclusive use of trans-editing domains prevents proline mistranslation. J Biol Chem 288:14391-9|
|Kumar, Sandeep; Das, Mom; Hadad, Christopher M et al. (2012) Substrate specificity of bacterial prolyl-tRNA synthetase editing domain is controlled by a tunable hydrophobic pocket. J Biol Chem 287:3175-84|
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