Scientific Report 2008
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
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
|Fig.1. Effects of mistranslation.
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.
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.
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.
Crystal structure of human glycyl-tRNA synthetase homodimer.
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
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