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Scientific Report 2008

Molecular Biology

Development of the Genetic Code and Its Connection to Human Disease

P. Schimmel, X.-L. Yang, R. Belani, E. Chong, M. Guo, R.T. Guo, M. Hanan, W.-W. He, I.L. Jung, M. Kapoor, J. Liu, E. Merriman, M.H. Nawaz, R. Shapiro, M. Vo, W. Zhang, Q. Zhou

We focus on aminoacyl tRNA synthetases and how their role as catalysts of aminoacylation is connected to disease and to broader biological systems through expanded functions that were developed over the long evolutionary history of the enzymes. The enzymes are ancient and are thought to have appeared in the transition from the putative RNA world to the theater of proteins. By linking amino acids to tRNAs that bear anticodon triplets encoding the attached amino acids, the synthetases establish the algorithm of the genetic code. Thus, alanyl-tRNA synthetases catalyze formation of Ala-tRNAAla, glycyl-tRNA synthetase catalyzes production of Gly-tRNAGly, and isoleucyl-tRNA synthetase produces Ile-tRNAIle. Altogether there are 20 tRNA synthetases, 1 for each amino acid, and the tRNA to which the amino acid is attached bears the triplet of the code for that amino acid. It is, therefore, this reaction that establishes the code.

Recent research established that heritable mutations in these 20 enzymes are causally linked to specific human diseases. This linkage occurs in one of two ways. First is the connection to mistranslation (Fig. 1). This connection occurs because many of the enzymes have an editing activity (encoded by a distinct active site) that clears amino acids when the amino acids are attached to the wrong tRNA. For example, alanyl-tRNA synthetase occasionally confuses glycine or serine for alanine, so Gly-tRNAAla or Ser-tRNAAla is produced. However, the occasional mischarged tRNA is cleared by a specific editing activity that can distinguish serine and glycine from alanine in the context of tRNAAla. Were these mischarged amino acids not cleared away, mistranslation would occur.

Fig.1. Effects of mistranslation.

Recently, we found that disruption of the editing activity not only is toxic to bacteria but also leads to serious pathologic changes in mammalian cells, in a trans-dominant way. Even a mild mutation, which produces only a 2-fold decrease in the activity for editing, leads to a heritable condition in mice, characterized by ataxia and neurodegeneration. We also discovered that mistranslation is mutagenic in aging bacteria. We are now exploring the possibility that mistranslation is mutagenic in mammalian cells in a way that could lead to oncogenic transformation.

How does alanyl-tRNA synthetase distinguish mischarged Ser-tRNAAla from the correctly charged Ser-tRNASer; that is, how does it use the context of the tRNA to strip serine from tRNAAla but not from tRNASer? The enzyme has distinct domains for aminoacylation (formation of charged tRNAAla) and editing (clearing of mischarged tRNAAla). These domains are encoded by polypeptides arranged in tandem along the sequence of alanyl-tRNA synthetase. For aminoacylation, tRNAAla synthetases throughout evolution are marked for aminoacylation with alanine by a single guanine-uracil base pair (G3:U70; located in the acceptor stem; Fig. 2). Transfer of this base pair into non-tRNAAla synthetases converts the enzymes into alanine-accepting tRNAs. The recognition of a single G3:U70 base pair is through determinants in the N-terminal aminoacylation domain of the protein. Remarkably, and to our surprise, this same base pair is used by the editing domain to pick out mischarged tRNAAla. Thus, the same base pair is recognized by distinct domains within the same protein.

Fig. 2. tRNAAla acceptor stems.

Last, in the human and mouse genomes, in addition to being encoded as part of alanyl-tRNA synthetase, the editing domain is separately encoded as a stand-alone fragment known as AlaXp. This fragment can also clear mischarged tRNAAla (Fig. 3). Thus, mammalian cells have developed 3 ways to prevent confusion of serine and glycine for alanine: (1) reasonably accurate (about 99%) selection of alanine in the aminoacylation step, (2) clearance of occasional errors, of confusing serine or glycine for alanine, by the editing domain of the enzyme, and (3) clearance of any residual mischarged tRNAAla by the free-standing AlaXp.

Fig. 3. Alanyl-tRNA synthetase (AlaRS) and artificial and natural editing-proficient fragments.

In summary, we started with the observation of how a single mutation in the site for editing led to disease in mice. From there, we uncovered the first example of 2 distinct domains in the same protein being able to recognize the same base pair.

The second connection of tRNA synthetases to heritable human diseases is through the expanded functions of the enzymes. Many human tRNA synthetases have functions beyond aminoacylation. For example, when activated, tyrosyl-tRNA synthetase and tryptophanyl-tRNA synthetase have opposing activities in angiogenesis. Lysyl-tRNA synthetase is incorporated into HIV virions for bringing in tRNALys, which is used as a primer for reverse transcription of the viral RNA genome during infection of a host cell. These extratranslation activities link aminoacyl-tRNA synthetases with boarder biological systems.

Two specific tRNA synthetases, tyrosyl- and glycyl-tRNA synthetase, are linked to Charcot-Marie-Tooth (CMT) disease, the most common heritable peripheral neuropathy; 1 of 2000 persons in the United States and Europe has the disease. Eleven different mutant alleles of GARS, the gene that encodes glycyl-tRNA synthetase, are reported to cause an axonal form of CMT in a dominant way. About half of the 11 CMT-causing mutants of glycyl-tRNA synthetase are unaffected in their aminoacylation activities. Similarly, dominant mutations in YARS, the gene that encodes tyrosyl-tRNA synthetase, have also been detected as the genetic cause of the disease in patients with CMT. Again, the mutations are not correlated with an aminoacylation defect. The lack of correlation of CMT mutations with aminoacylation activity suggests additional, expanded functions for glycyl- and tyrosyl-tRNA synthetases in neuronal cells.

The 11 CMT-linked mutations are spread out on the primary sequence of glycyl-tRNA synthetase. However, as revealed in our crystal structure of the synthetase, all of the CMT-associated residues are located on or near the dimerization interface of the α2 homodimer (Fig. 4). Strikingly, 2 CMT-causing mutations, each from a different subunit, are associated with residues in the wild-type enzyme that make contact with each other across the dimer interface. This finding suggests that the dimer interface is critical for the potential expanded function of glycyl-tRNA synthetase. Because both monomer and dimer forms of the synthetase coexist in equilibrium, we hypothesized that the monomer form, which is inactive for aminoacylation, has a distinct biological role specific for neuronal cells. Disruption of this expanded role links glycyl-tRNA synthetase to the etiology of CMT.

Fig. 4. Crystal structure of human glycyl-tRNA synthetase homodimer.

Interestingly, when transfected into a mouse neuroblastoma N2a cell line, genes encoding each of the CMT-causing glycyl-tRNA synthetase mutants were defective in distribution into neurite terminals. This distribution defect is reminiscent of the muscle weakness around the terminal nerves in patients with CMT. We have speculated that the CMT-associated mutations of glycyl-tRNA synthetase affect transportation of the synthetase into the neurites via a mechanism linked to the dimerization interface.

Our awareness of the connections of tRNA synthetases, which are intimately associated with the development of the genetic code, to diseases is only beginning. We anticipate that many more connections will be found.


Bacher, J., Waas, W.F., Metzgar, D., de Crécy-Lagard, V., Schimmel, P. Genetic code ambiguity confers a selective advantage on Acinetobacter baylyi. J. Bacteriol. 189:6494, 2007.

Beebe, K., Mock, M., Merriman, E., Schimmel, P. Distinct domains of tRNA synthetase recognize the same base pair. Nature 451:90, 2008.

Beebe, K., Waas, W., Druzina, Z., Guo, M., Schimmel, P. A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities. Anal. Biochem. 368:111, 2007.

Greenberg, Y., King, M., Kiosses, W.B., Ewalt, K., Yang, X.-L., Schimmel, P., Reader, J.S., Tzima, E. The novel fragment of tyrosyl-tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells. FASEB J. 22:1597, 2008.

Guo, M., Ignatov, M., Musier-Forsyth, K., Schimmel, P., Yang, X.-L. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: implications for multisynthetase complex formation. Proc. Natl. Acad. Sci. U. S. A. 105:2331, 2008.

Kapoor, M., Zhou, Q., Otero, F., Myers, C.A., Bates, A., Belani, R., Liu, J., Luo, J.-K., Tzima, E., Zhang, D.-E., Yang, X.-L., Schimmel, P. Evidence for annexin II-S100A10 complex and plasmin in mobilization of cytokine activity of human TrpRS. J. Biol. Chem. 283:2070, 2008.

Waas, W.F., Druzina, Z., Hanan, M., Schimmel, P. Role of a tRNA base modification and its precursors in frameshifting in eukaryotes. J. Biol. Chem. 282:26026, 2007.

Waas, W.F., Schimmel, P. Evidence that tRNA synthetase-directed proton transfer stops mistranslation. Biochemistry 46:12062, 2007.

Yang, X.-L., Kapoor, M., Otero, F.J., Slike, B.M., Tsuruta, H., Frausto, R., Bates, A., Ewalt, K.L., Cheresh, D.A., Schimmel, P. Gain-of-function mutational activation of a human tRNA synthetase procytokine. Chem. Biol. 14:1323, 2007.

Zhou, Q., Kiosses, W.B., Liu, J., Schimmel, P. Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine. Methods 44:190, 2008.


Paul R. Schimmel, Ph.D.
Ernest and Jean Hahn Professor of Molecular Biology and Chemistry

Xiang-Lei Yang, Ph.D.
Assistant Professor of Molecular Biology

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