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Genetic code ambiguity: an unexpected source of proteome
innovation and phenotypic diversity
Gabriela R Moura, Laura C Carreto and Manuel AS Santos
Translation of the genome into the proteome is a highly
accurate biological process. However, the molecular
mechanisms involved in protein synthesis are not error free and
downstream protein quality control systems are needed to
counteract the negative effects of translational errors
(mistranslation) on proteome and cell homeostasis. This plus
human and mice diseases caused by translational error
generalized the idea that codon ambiguity is detrimental to life.
Here we depart from this classical view of deleterious
translational error and highlight how codon ambiguity can play
important roles in the evolution of novel proteins. We also
explain how tRNA mischarging can be relevant for the synthesis
of functional proteomes, how codon ambiguity generates
phenotypic and genetic diversity and how advantageous
phenotypes can be selected, fixed, and inherited. A brief
introduction to the molecular nature of translational error is
provided; however, detailed information on the mechanistic
aspects of mistranslation or comprehensive literature reviews
of this topic should be obtained elsewhere.
Address
Department of Biology and CESAM, University of Aveiro, 3810-193
Aveiro, Portugal
Corresponding author: Santos, Manuel AS ([email protected])
Current Opinion in Microbiology 2009, 12:1–7
This review comes from a themed issue on
Growth and development: eukaryotes
Edited by Judith Berman
1369-5274/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2009.09.004
Introduction
Genome translation errors can arise during tRNA charging by aminoacyl-tRNA synthetases (aaRSs) and during
mRNA decoding by the ribosome. Aminoacylation errors
are mainly caused either by failure of the aaRSs to
differentiate between amino acids with similar chemical
properties or by the incorrect recognition of tRNAs. Such
errors are minimized by aaRSs editing mechanisms,
which discard incorrectly bound amino acids, and by
highly specific tRNA–aaRS interaction networks [1].
At the ribosome level, mRNA decoding is affected by
four main types of errors: first, missense errors cause
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incorrect amino acid incorporation into growing polypeptide chains and result in the synthesis of mutant proteins;
second, nonsense errors cause readthrough of stop codons
and produce proteins with extended C-termini; third,
frameshifting errors alter the mRNA reading frame normally to the 1 or to the +1 frames, producing out-offrame truncated proteins; and fourth, processivity errors
terminate translation prematurely and also produce truncated proteins (Figure 1) [2].
On average, the frequency of translational errors is in the
order of 104 [1]. These are regarded as physiological
errors and quality control systems minimize the toxic
effects of mistranslated proteins. However, the fidelity
of protein synthesis is influenced by intracellular and
extracellular factors and fluctuates over time and along
mRNA reading frames. Translation errors increase sharply under amino acid starvation and are affected by codon
usage, codon context, mRNA structure, aminoacyl-tRNA
concentration, and tRNA modification, which in turn are
influenced by cellular metabolism [3,4–7]. Also, in yeast
and probably in all eukaryotes, translation termination
fidelity is controlled by the [PSI+] factor that is a prion
form of the eukaryotic translation termination factor 3
(eRF3) [8,9]. Environmental conditions that promote
[PSI+] formation increase suppression of stop codons
and synthesis of proteins with extended C-termini [10].
Therefore, translational error is dynamic and can be
regulated. We discuss below how natural selection
explored mistranslation to expand the genetic code and
to create novel functional classes of proteins. We also
discuss the evolutionary implications of recent studies
showing that codon ambiguity generates phenotypic
variability and genetic diversity. For simplicity, throughout the text, translational error, mistranslation, genetic
code ambiguity, and codon ambiguity have identical
meaning.
tRNA mischarging as a source of proteome
innovation
The evolutionary relevance of genetic code ambiguity is
unequivocally demonstrated by the natural reassignments
of UGA and UAG stop codons to selenocysteine (Sec) and
pyrrolysine (Pyl), respectively, and by the widespread
tRNA-dependent synthesis of asparagine (Asn), glutamine (Gln), and cysteine (Cys) [11,12,13], as discussed
below.
Selenocysteine (Sec) replaces cysteine (Cys) in the active
site of a group of redox proteins generically known as
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Please cite this article in press as: Moura GR, et al. Genetic code ambiguity: an unexpected source of proteome innovation and phenotypic diversity, Curr Opin Microbiol (2009), doi:10.1016/
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2 Growth and development: eukaryotes
Figure 1
Sources of genetic code ambiguity during genome translation into the proteome. Codons may be misassigned to amino acids due to tRNA
mischarging by aminoacyl-tRNA synthetases (aaRSs) or due to codon misreading during mRNA decoding in the ribosome. Mischarging of tRNAs may
be caused by the failure of aaRSs to recognize their cognate tRNAs or by the activation of incorrectly bound amino acids. To minimize mischarging,
some aaRSs have editing mechanisms that discard chemically similar amino acids from their active sites. In some organisms, tRNA mischarging is
necessary for the synthesis of selenocysteine, asparagine, glutamine, and cysteine. Codon decoding errors are mainly caused by misreading of sense
codons (missense errors) and nonsense codons (nonsense errors) by near cognate or noncognate tRNAs. These errors result in the synthesis of
mutant proteins. Other mRNA decoding errors are caused by loss of the reading frame (frameshifting) or premature ribosome drop off from the mRNA
(processivity errors). These errors result in premature translation termination and synthesis of truncated polypeptides.
selenoproteins. Sec is translationally inserted at specific
UGA codons which are reprogramed by a complex translational machinery called the selenosome [11,14]. Initial
charging of a unique tRNASec with serine (Ser) by a seryltRNA synthetase (SerRS) produces a mischarged tRNA
species (Ser-tRNASec) whose Ser moiety is then converted to selenocysteyl-tRNASec [Sec-tRNASec] by a
selenocysteine synthase (SelA), using selenophosphate
as substrate [12]. The existence of the mischarged SertRNASec suggests, therefore, that Ser can be misincorporated at Sec-UGA codons, creating codon ambiguity.
Expression of the Eubacterium acidaminophilum peroxiredoxin PrxU gene in Escherichia coli results in the misincorporation of selenocysteine, tryptophan, and serine at
the UGA-Sec codons [14], confirming that hypothesis.
In the ciliate Euplotes crassus cysteine and selenocysteine
are naturally inserted at UGA codons [15]. This ciliate
genome encodes two tRNAs with cognate anticodons for
the UGA codon, namely the tRNAUCASec and the tRNAUCys
. Its thioredoxin reductase genes (eTR1 and eTR2)
CA
contain seven UGAs, the first six direct insertion of Cys
while the last one is used to insert Sec. Cys is incorporated
by the tRNAUCACys because of failure of the E. crassus
eRF1 to recognize the UGA stop codon and Sec is
Current Opinion in Microbiology 2009, 12:1–7
incorporated by the tRNAUCASec through recoding of
the UGA by a SECIS element present in the 30 -UTR
of eTR1 and eTR2. Cys and Sec incorporation at those
UGAs are necessary to produce active eTR1 and eTR2
[15], thus demonstrating that such ambiguity is functional.
Pyrrolysine (Pyl) provides additional evidence for positive
roles of genetic code ambiguity. Pyl is cotranslationally
inserted into the active center of methyltransferases of
Methanosarcineace species, Desulfitobacterium hafniense, and
in the glutless worm Olavius algarvensis [16]. Pyl is charged
on a dedicated nonsense suppressor tRNA (tRNAPyl) by a
pyrrolysyl-tRNA synthetase (PylRS) that catalyzes the
formation of Pyl-tRNAPyl [12]. Surprisingly, the PylRS
has broad amino acid specificity and binds various nonnatural lysine derivatives, namely Ne-D-prolyl-L-lysine, Necyclopentyloxycarbonyl-L-lysine (Cyc), Ne-(tert-butoxycarbonyl (t-Boc))-L-lysine (Boc-lysine), Ne-t-Boc-L-6-amino2-hydroxyhexanoic acid (Boc-LysOH) [17,18]. These
amino acids are mischarged onto the tRNAPyl by PylRS
and can be inserted into the genetic code of E. coli, yeast, or
mammalian cells using the orthogonal PylRS.tRNAPyl pair
[18,19,20]. Furthermore, the Methanosarcina barkeri
Fusaro tRNAPyl can be charged with serine in vitro by
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Mistranslation generates phenotypic diversity Moura, Carreto and Santos 3
one of its SerRS enzymes and in vivo by the E. coli SerRS
[19], that is, it can exist as Pyl-tRNAPyl or Ser-tRNAPyl.
This broad amino acid specificity of the PylRS and ancestral competition of the tRNAPyl with release factor 1 (RF1)
for UAG codons strongly suggest that the ancestral UAGPyl was ambiguous and that its recoding evolved to minimize such ambiguity.
Other cases of natural tRNA ambiguity are illustrated by
tRNA-dependent asparagine (Asn), glutamine (Gln), and
cysteine (Cys) biosynthesis in various organisms where
AsnRS, GlnRS, and CysRS do not exist (reviewed in
[13]). In most bacteria and archaea Asn is synthesized
directly on a tRNAAsn which is initially charged with Asp
by a nondiscriminating AspRS (Asp-tRNAAsn). In all
known archaea, most bacteria and chloroplasts, Gln is
synthesized on a tRNAGln that is mischarged with Glu by
a nondiscriminating GluRS (Glu-tRNAGln) [21,22].
Amido transferases (Glu-AdT and Asp-AdT) carry out
the conversion of Asp and Glu into Asn and Gln and also
phosphorylate the mischarged tRNAs producing two
additional mischarged intermediate tRNA species,
namely g-phosphoryl-Glu-tRNAGln (P-Glu-tRNAGln)
(P-Asp-tRNAAsn)
and
b-phosphoryl-Asp-tRNAAsn
[23,24]. Similarly, in most methanogenic archaea,
cysteine (Cys) is synthesized on a tRNACys that is initially
charged with O-phosphoserine (Sep) by the enzyme Ophosphoseryl-tRNA synthase (SepRS) (Sep-tRNACys). A
Sep-tRNA:Cys-tRNA synthase (SepCysS) then transforms Sep-tRNACys into Cys-tRNACys [25,26,27,28].
Considering that mischarged tRNA can participate in
mRNA translation [29,30,31], it is reasonable to assume
that Asn, Gln, and Cys were also incorporated into the
genetic code of the ancestor of modern organisms through
ambiguous translation of the respective codons. It is not
yet clear why Asn, Gln, and Cys are synthesized directly
on mischarged tRNAs in these organisms, but these
alternative biosynthetic pathways may integrate amino
acid metabolism, protein synthesis, and cellular signaling
pathways providing new layers of cellular control [13].
Codon ambiguity as a generator of phenotypic
diversity
In several species of the genus Candida thousands of
leucine CUG codons have been reassigned to serine using
a novel serine tRNA (tRNACAGSer) [30,32]. These CUG
codons remain ambiguous in extant Candida species due
to charging of the tRNACAGSer by both the SerRS and the
LeuRS [33]. This leads to the formation of a correctly
charged Ser-tRNACAGSer and a mischarged Leu-tRNASer
which participate in translation. Under laboratory
CAG
conditions, serine and leucine are incorporated into the
Figure 2
The consequences of genetic code ambiguity. Codon ambiguity results in the synthesis of mutant proteins, which misfold and are targeted for
degradation by the ubiquitin–proteasome quality control system. Alternatively, such proteins aggregate and accumulate in the cell forming toxic
protein aggregates. Some mutant proteins may still fold with the help of molecular chaperones and may retain their functions or may acquire new
functions. Accumulation of aggregated and soluble mutant proteins and overloading of protein quality control systems with misfolded proteins activate
the stress response and reprogram gene expression, leading to exposure of hidden phenotypic variation. Mutant DNA polymerases and DNA repair
enzymes create hypermutagenic clones with high adaptation potential. Under these genetic code ambiguity conditions, cell viability, and homeostasis
are guarantied by wild type proteins that represent the major component of the proteome.
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4 Growth and development: eukaryotes
Figure 3
Model for fixation and inheritance of phenotypes associated with genetic code ambiguity. Ambiguous codon decoding expands the proteome and
generates new phenotypes creating phenotypic diversity. Selection of advantageous phenotypes creates positive feedback pressure that maintains
ambiguous codon decoding. This ultimately leads to synthesis of mutant DNA polymerases and DNA repair enzymes and emergence of
hypermutagenic clones and to an exponential increase in genome mutational load. This accelerates the fixation of advantageous phenotypes and their
transmission to the progeny.
Candida albicans proteome with efficiencies of 97% and
3%, respectively, but such CUG ambiguity increases up
to 5% under stress. C. albicans tolerates up to 28% of
leucine misincorporation without visible effects on
growth rate, suggesting that CUG ambiguity may fluctuate between 3% and 28%, depending on environmental
conditions. Remarkably, CUG ambiguity increases
secretion of lipases and proteinases and cell adhesion
and spawns a wide variety of colony morphologies
[34,35]. In S. cerevisiae, similar CUG ambiguity does
not generate such phenotypic and morphological diversity; however, it increases tolerance to various environmental stressors and permits growth in the presence of
lethal doses of cycloheximide, arsenite, cadmium, and salt
[36]. Most interestingly, the phenotypic diversity
spawned by CUG ambiguity in C. albicans is similar to
that spawned in S. cerevisiae by the [PSI+] prion [37]. The
[PSI+] prion reduces decoding efficiency of the translation termination machinery (eRF1–eRF3 complex)
leading to readthrough of UAG, UAA, and UGA stop
codons and to synthesis of proteins with extended Ctermini. Accumulation of such aberrant proteins spawns a
wide variety of growth and morphological phenotypes
with high selective potential [10]. [PSI+] also controls the
biosynthesis of polyamines by regulating the expression
of ornithine decarboxilase antizyme through a +1 frameshift at a shifty-UGA site [38]. Therefore, [PSI+]
mediated stop codon ambiguity in S. cerevisiae and
CUG ambiguity in C. albicans are used as sources of
phenotypic variation and for novel regulatory mechanisms (Figure 2).
Generation of genetic diversity and
inheritance of phenotypic traits
An important question related to the phenotypic diversity
that arises from genetic code ambiguity is how can it be
Current Opinion in Microbiology 2009, 12:1–7
selected and inherited? Clearly, advantageous phenotypes exposed by codon ambiguity can only be evolutionarily relevant if transmitted to the progeny. There are
two fundamental difficulties here. Firstly, the phenotypic
diversity generated by codon ambiguity is diverse and
somewhat stochastic. Secondly, in general, codon ambiguity decreases fitness and should be eliminated by
negative selection. The discovery in E. coli and other
bacteria of a hypermutagenesis phenotype associated
with codon ambiguity, the ‘translational stress mutagenesis phenotype’ (TSM), provides a fascinating solution for
this apparent genetic paradox [39–43]. A mutant tRNAGly
that misincorporates glycine at aspartate (GAC/U)
codons, various mutant tRNAAla that misread noncognate
codons, ambiguous ribosomes, and the mistranslation
drug streptomycin all induce spontaneous hypermutagenesis in E. coli via synthesis of mutant DNA polymerase III
and RecABC/RuvABC complexes [39,40,41]. This
increased genome mutation rate generates genetic diversity and, therefore, provides an indirect mechanism for
rapid fixation of the advantageous phenotypes linked to
genetic code ambiguity (Figure 3).
Conclusions
The view that codon ambiguity is an aberration of nature
is clearly a poor interpretation of the biology of this old
phenomenon. There is no doubt that above a certain
mistranslation threshold the proteome is disrupted, cell
fitness decreases, and cell death increases [31,44,45]. But,
codon ambiguity can increase proteome diversity by
creating statistical populations of proteins and can generate genetic and phenotypic diversity, which can be
explored by natural selection for developmental, metabolic, and regulatory innovation. It is now clear that
bacteria and eukaryotes are far more resistant to mistranslation than anticipated and that mischarged tRNAs can
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Mistranslation generates phenotypic diversity Moura, Carreto and Santos 5
Table 1
Some known cases of functional genetic code ambiguity
Function
Organisms
Reference
Natural ambiguity
Ser-tRNA Sec
Asp-tRNA Asn
P-Asp-tRNA Asn
Glu-tRNA Gln
P-Glu-tRNA Gln
Sep-tRNA Cys
Leu-tRNA Ser
PSI prion
Selenocysteine incorporation
Asp synthesis
Asp synthesis
Gln synthesis
Gln synthesis
Cys synthesis
Leu misincorporation
Readthrough of stop codons
Most prokaryotes and eukaryotes
Most bacteria, archaea, and mitochondria
Most bacteria, archaea, and mitochondria
Most bacteria, archaea, and mitochondria
Most bacteria, archaea, and mitochondria
Most bacteria, archaea, and mitochondria
Various Candida species
Yeast and other eukaryotes
[11,12]
[13]
[13]
[13]
[13]
[13]
[32,33]
[37]
Artificial ambiguity
aa*-tRNA–aaRS
Orthogonal pairs
Cys-tRNAPro
Ser-tRNAThr
X*-tRNA Pyl
Genetic code expansion
Mistranslation
Mistranslation
Genetic code expansion
Escherichia coli, yeast, and mammalian cells
E. coli
E. coli
E. coli
[50]
[29]
[29]
[19]
Ambiguity at the tRNA charging level is not always translated into proteins due to translational buffering systems, namely the capacity of the
elongation factor Tu (EF-Tu) to negatively discriminate mischarged tRNAs. However, naturally mischarged tRNAs should have participated in
translation before such buffering systems appeared. This is corroborated by lack of such buffering systems in E. coli, yeast, and mammalian cells
engineered for the incorporation of both artificial and natural amino acids through orthogonal tRNA–aaRS pairs [49,50]. X* indicates artificial
derivatives of lysine.
participate in mRNA translation (Table 1) [29,46].
The high cellular tolerance to mistranslation opens the
possibility for the evolution of proteomic and phenotypic
novelty, which can be fixed rapidly through hypermutagenesis. In other words, genetic code ambiguity is an
epigenetic evolution evolvability system much similar to
[PSI+] or Hsp90 [47,48,49].
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Acknowledgements
We are thankful to the Portuguese Foundation for Science and Technology
and to the Human Frontier Science Program for funding our work through
projects PTDC/BIA-BCM/64745 and RGP0045/2005-C, respectively. We
are also grateful to Dieter Söll and Judith Berman for reading and
commenting on the manuscript before publication.
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Please cite this article in press as: Moura GR, et al. Genetic code ambiguity: an unexpected source of proteome innovation and phenotypic diversity, Curr Opin Microbiol (2009), doi:10.1016/
j.mib.2009.09.004