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The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases

Raina, Medha Vijay
http://rave.ohiolink.edu/etdc/view?acc_num=osu1398856775

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Aminoacyl-tRNA synthetases (aaRSs) catalyze the first step of translation, aminoacylation. These enzymes attach amino acids (aa) to their cognate tRNAs to form aminoacyl-tRNA (aa-tRNA), an important substrate in protein synthesis, which is delivered to the ribosome as a ternary complex with translation elongation factor 1A (EF1A) and GTP. All aaRSs have an aminoacylation domain, which is the active site that recognizes the specific amino acid, ATP, and the 3' end of the bound tRNA to catalyze the aminoacylation reaction. Apart from the aminoacylation domain, some aaRSs have evolved additional domains that are involved in interacting with other proteins, recognizing and binding the tRNA anticodon, and editing misacylated tRNA thereby expanding their role in and beyond translation. One such function of the aaRS is to form a variety of complexes with each other and with other factors by interacting via additional N or C terminal extensions. For example, several archaeal and eukaryotic aaRSs are known to associate with EF1A or other aaRSs forming higher order complexes, although the role of these multi-synthetase complexes (MSC) in translation remains largely unknown. MSC function was hence investigated in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aaRS activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These in vivo data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies and could potentially increase translation efficiency by limiting substrate diffusion from the ribosome, thus facilitating rapid recycling of tRNAs. In addition to their aminoacylation functions, about half of the aaRSs have evolved an editing function, which hydrolyzes non-cognate amino acid from its cognate tRNA thereby maintaining the fidelity of translation. Phenylalanyl-tRNA synthetase (PheRS) misactivates and mischarges Tyr onto tRNAPhe, but is able to correct the mistake using a proofreading activity which hydrolyzes mischarged Tyr-tRNAPhe. The requirement for PheRS editing and fidelity of Phe codon translation is specific for different cellular compartments in eukaryotes and varies significantly within bacteria. Yeast cytoplasmic PheRS (ctPheRS) has a low Phe/Tyr specificity and is capable of editing, whereas the yeast mitochondrial enzyme (mtPheRS) completely lacks an editing domain, and instead relies on high Phe/Tyr specificity. Escherichia coli, in contrast, has retained features of both yeast enzymes and displays a high degree of Phe/Tyr specificity and robust editing activity. We showed that in E. coli the editing domain has evolved to efficiently edit m-Tyr-tRNAPhe, and that this editing activity is essential for cellular growth and viability in the presence of the non-proteinogenic amino acid m- Tyr and oxidative stress conditions. In comparison, in the yeast enzyme, due to the low specificity of its active site, cytoplasmic PheRS editing has evolved to protect the proteome from p-Tyr misincorporation as shown by the requirement for editing activity to survive in the presence of high concentrations of Tyr compared to Phe. Hence different environmental factors and cell physiology drive the selection of quality control mechanisms in various organisms. While PheRS has evolved to possess editing activity to actively edit noncognate amino acids (both proteinogenic and non-proteinogenic), editing mechanisms are not evolutionarily conserved. Tyrosyl-tRNA synthetases are among the aaRSs lacking any known editing activity. The high specificity displayed by this aaRSs is achieved by taking advantage of the unique structural and chemical properties of certain amino acids, leading to favorable binding affinities of cognate over non-cognate substrates in the active site of the enzyme. Its cognate amino acid Tyr differs from Phe by a single hydroxyl group, and the specific recognition and binding of the hydroxyl group allows bacterial TyrRS to discriminate against non-cognate Phe with a specificity of 105. However, recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. We examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with the decrease of Tyr in the amino acid pools, suggesting that mischarging of tRNATyr with non-cognate Phe by TyrRS was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic TyrRS revealed a twenty five-fold lower specificity for Tyr over Phe compared to previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial TyrRSs revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.
Michael Ibba, Dr. (Advisor)
Juan Alfonzo, Dr. (Committee Member)
Irina Artsimovitch, Dr. (Committee Member)
Kurt Fredrick, Dr. (Committee Member)
Karin Musier-Forsyth, Dr. (Committee Member)
206 p.
Biochemistry
protein synthesis; tRNA; aminoacyl-tRNA synthetase; multi-synthetase complex; quality control; editing

Recommended Citations

Citations

  • Raina, M. V. (2014). The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398856775

    APA Style (7th edition)

  • Raina, Medha. The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1398856775.

    MLA Style (8th edition)

  • Raina, Medha. "The Role in Translation of Editing and Multi-Synthetase Complex Formation by Aminoacyl-tRNA Synthetases." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398856775

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.