YeastYeast 2006; 23: 203–213.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1350

Review

Evolution of the genetic code in yeastsIsabel Miranda, Raquel Silva and Manuel A. S. Santos*Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

*Correspondence to:Manuel A. S. Santos, Centre forCell Biology, Department ofBiology, University of Aveiro,3810-193 Aveiro, Portugal.E-mail: msantos@bio.ua.pt

Received: 23 December 2005Accepted: 28 December 2005

AbstractDuring the last 30 years, a number of genetic code alterations have been uncovered inbacteria and in the mitochondria and cytoplasm of various eukaryotes, invalidatingthe hypothesis that the genetic code is universal and frozen. In the mitochondriaof most yeasts, the UGA stop codon is decoded as tryptophan and the four leucinecodons of the CUN family (N = any nucleotide) are decoded as threonine. Recently, aunique genetic code change involving the decoding of the leucine CUG codon as serinewas discovered in the cytoplasm of Candida and Debaryomyces species, indicatingthat the genetic code of yeasts may be under specific evolutionary pressures whosemolecular nature is not yet fully understood. This genetic code alteration is mediatedby a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (ser-tRNACAG)through insertion of an adenosine in the intron of its gene. This event, which occurred272 ± 25 million years ago, reprogrammed the identity of approximately 30 000 CUGcodons existent in the ancestor of these yeasts and had a profound impact on theevolution of the genus Candida and of other species. Here, we review the most recentresults and concepts arising from the study of this genetic code change and highlighthow its study is changing our views of the evolution of the genetic code. Copyright 2006 John Wiley & Sons, Ltd.

Keywords: genetic code; codon reassignment; tRNAs; amino acy-tRNA synthetases;protein synthesis

Contents

IntroductionMolecular theories for the evolution ofgenetic code alterationsEvolution of genetic code alterations in yeastTime scale of CUG reassignmentOther implications of CUG reassignmentConclusionsReferences

Introduction

To date, 10 alterations to the standard genetic codehave been found in the cytoplasm of both prokary-otes and eukaryotes and 16 in the mitochondria ofseveral organisms ranging from yeasts to humans[10]. In mitochondria, the UAG stop codon is reas-signed to Ala or Leu, the UAA stop is reassignedto Tyr and the UGA to Trp. The arginine AGR

(R = G or A) codons are often reassigned to Ser,Gly or stop, the AUA-Ile codon is sometimes reas-signed to Met, while the AAA-Lys is reassigned toAsn and other codons are not used (unassigned) inthe open reading frames (ORFeome). In bacteria,and in the cytoplasm of eukaryotes, genetic codechanges involve reassignment of nonsense codonsand are a subset of those found in mitochondria.In various ciliates, the UAG and UAA stop codonsare reassigned to glutamine and the UGA to Trp orCys. In Mycoplasma capricolum the CGG codonand the Micrococcus luteus AGA and AUA codonsare unassigned (Figure 1).

The genetic codes of various yeasts have a num-ber of deviations from the standard code. The mito-chondrial UGA-stop codon is decoded as Trp inmost yeasts, the four codons of the CUN-Leu fam-ily are decoded as Thr in the mitochondria of Sac-charomyces cerevisiae and Candida glabrata, andin the cytoplasm of various ascomycetes, including

Copyright 2006 John Wiley & Sons, Ltd.

204 I. Miranda, R. Silva and M. A. S. Santos

Key:Mitochondria onlyYeasts citoplasm and mitochondriaSome bacteria, eukaryotes andmitochondria

Stop; Selenocysteine;

Cys; Trp

A CG T

AA

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luC

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Stop; Gln; pyrrolysine

Figure 1. Diversity of the genetic code. To date a number of genetic code changes have been uncovered in eubacteria,archaea and in the eukaryotic cytoplasm and mitochondria. In mitochondrial systems, both sense and nonsense codonscan change identity but in prokaryotic and eukaryotic nuclear systems only stop codons change identities, the exceptionbeing the decoding of the leucine-CUG codon as serine in Candida spp. (red). The prokaryotic and eukaryotic nucleargenetic code changes are a subset of those found in mitochondria, indicating that there are codon sets that are moreprone to changing their identity than others. In particular, codons starting with T or A change their identity rather often,while codons starting with C only change their identity in yeast mitochondria and in Candida, where the leucine CUNcodon family is decoded as threonine and the CUG codon is decoded as serine, respectively. Codons starting with G areapparently resistant to identity changes, suggesting that the strength of codon–anticodon interaction at the first codonposition is an important determinant of the evolution of non-standard genetic codes

species of the genus Candida and Debaryomyces;the Leu CUG codon is decoded as Ser [14]. There-fore, the genetic code is still evolving, suggest-ing that reassigning a stop or a sense codon inan organelle with a small proteome may not besuch a complicated task. The rarity of the bacterialand eukaryotic cytoplasmic genetic code changessuggests, however, that reassigning a codon in anorganism with a large proteome may be much morecomplex. The Candida genetic code alteration andthe recent artificial expansion of the genetic codein Escherichia coli and yeast show that the codehas intrinsic flexibility and is amenable to manipu-lation by genetic engineering and synthetic biologymethodologies [5,6]. Despite this, the molecularmechanisms that mediate genetic code alterationsand their impact on physiology, genome evolutionand gene expression remain an almost completemystery. Some progress has been made on theformulation of theories that provide models for the

evolutionary pathways of genetic code alterations.These theories are reviewed here to provide thereader with a theoretical framework for understand-ing how genetic code alterations may evolve, andto highlight the importance of yeasts as model sys-tems for studying the evolution of the genetic code.

Molecular theories for the evolution ofgenetic code alterations

The codon capture theory (neutral model[14,15])

This theory postulates that certain codons disap-pear from the coding component of a genome(ORFeome) under biased genome A + T or G + Cpressure (Figure 2A, B). These vanished codons(unassigned codons) may be reintroduced by gene-tic drift and upon re-emergence they may be reas-signed by misreading tRNAs of non-cognate amino

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 203–213.

Yeasts genetic code 205

acid families. The impact of this event on theproteome is nil. This theory is supported by thedisappearance of the CGG codon in Mycoplasmacapricolum (25% genome G + C) [9,16] and theAGA and AUA codons in Micrococcus luteus(75% genome G + C) and postulates that geneticcode changes are a direct consequence of biasedgenome G + C pressure arising from mutationsin DNA polymerases or DNA repair systems(Figure 2A–C).

The reassignment of certain sense and nonsensecodons in organisms whose genomes do not displayany obvious G + C or A + T bias, and in caseswhere codon reassignment occurs against suchbias (e.g. the reassignment of the leucine CUUand CUA codons to threonine in the A + T rich

A1. Codons disappear under biasedAT or GC pressure

CGA ArgCGGArg

CGU Arg

2. Arg – tRNA CCG disappears

M. capricolum M. luteus%GC 25 74Unassigned CGG AGA, AUA

(arg) (arg ile)

CGG G CGG AAA AGA 3'

New strand CCT T CC TTT TCT

Second replication cycle

B

C

First replication cycle: high A+T pressure

CG A CGA CGAAAA AGA

Template

TCC

CG

Figure 2. Codon unassignment and capture through biasedgenome G + C pressure. (A) Strong G + C pressure arisingfrom mutations in DNA polymerases and/or DNA repairsystems alters codon usage significantly. In extreme cases,biased G + C pressure may lead to codon disappearancefrom the genome. (B) The disappearance of the arginineAGA and the isoleucine AUA codons from the genomeof Micrococcus luteus (74% G + C) and the arginine CGGcodon from the genome of Mycoplasma capricolum (25%G + C) provide strong support for this mechanism [14]. Asimilar mechanism is apparently in action in the A + T-richmitochondrial genomes, which display a rather high geneticcode diversity. In these cases, degeneracy of the geneticcode works as a buffer, permitting codon disappearancewithout changing protein sequence. Once a codon hasdisappeared from the genome, it can be reintroduced bygenetic drift, allowing for redefinition of the identity of thenewly introduced codon if a tRNA from a non-cognatecodon family misreads it. The existence of a misreadingtRNA (wild-type or mutant) is critical to ensure decodingof the reintroduced codon; if the codon is not decoded, itwill stall the ribosome during mRNA translation and blockprotein synthesis. The misreading tRNA can then acquirea cognate anticodon for the ‘new’ codon, which will allowfor its efficient decoding during mRNA translation and fullcapture of the reintroduced codon [14]

genome of yeast mitochondria), raised questions asto the validity of the ‘codon capture’ theory andprompted the development of alternative theoriesfor the evolution of genetic code alterations [23].This was further supported by the observation thatgenome G + C pressure is not evenly distributedalong eukaryotic chromosomes.

The ambiguous intermediate theory(selection-driven hypothesis [24,25])

This theory postulates that mutant tRNAs withdouble identity, which are recognized by morethan one aminoacyl-tRNA synthetase, or tRNAswith expanded decoding properties (misreadingtRNAs), may drive genetic code changes throughan ambiguous codon decoding mechanism. Inother words, ambiguous codon decoding pro-vides an initial step for gradual codon identitychange, and wild-type or mutant misreading tRNAsare the critical elements of codon reassignment(Figure 3A–C). Codon disappearance is not a pre-requisite of codon reassignment but wild-type cog-nate tRNAs must disappear from the genome for acomplete change of codon identity.

Codon decoding ambiguity is problematic, sinceit destabilizes the proteome and reduces fitness;however, the ‘ambiguous intermediate theory’ hasbeen given strong support by the discovery thatin certain Candida species the CUG codon isstill ambiguous, i.e. it is decoded as both serineand leucine, due to charging of the Ser-tRNACAGby both the seryl- and leucyl-tRNA synthetases(SerRS and LeuRS, respectively) [27]. Further-more, the Bacillus subtilis UGA stop codon isdecoded as both tryptophan or stop, and variousorganisms and viruses use the stop codons UGA,UAA or UAG in a programmed fashion to regu-late the expression of particular genes [2]. That is,ambiguous codon decoding is an intrinsic charac-teristic of mRNA translation, whether programmedor the result of random decoding error (standarderror rate = 10−4).

Is there a unifying model for codonreassignment?

The ‘codon capture’ and ‘ambiguous intermedi-ate’ theories are not mutually exclusive, sincebiased G + C content reduces codon usage or maydrive codons from the entire ORFeome [16]. Suchdecreased usage of a particular codon may, in turn,

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206 I. Miranda, R. Silva and M. A. S. Santos

CUG

Leu

CUG

Leu Ser

Leu

Disappears

A Candida cytoplasm

B Yeast mitochondriaAmbiguous

CUN

Leu

CUN

Leu Thr

MutantThr - tRNA

Leu

Disappears

Ambiguouscodons

NewThrRS

+

CUG

SerMutant

Ser-tRNA

CUN

Thr

Identityredefined

Identityredefined

UGA

Trp

UGA

Nonsensesuppressor

Trp Releasefactor

Ambiguouscodon

Identityaltered

Mutant mRF2disappears

from the genome

No recognitionof stop codon

C Yeast mitochondria

mRF2mRF2

Figure 3. Mechanisms of codon identity redefinition inyeasts. (A) CUG codon in the Candida cytoplasm. Theidentity of the ancestral CUG codon was redefined dueto the appearance of a mutant Ser-tRNA that decoded itas serine. Initially, the CUG codon was ambiguous, dueto competition between the cognate Leu-tRNA and themutant Ser-tRNA. The identity of the CUG codon wascompletely redefined upon disappearance of the Leu-tRNA,which decoded the codon according to the standardgenetic code rules. (B) Redefinition of the identity of theCUN codon family. In the mitochondria of S. cerevisiae,C. glabrata and other yeasts, the identity of the entireleucine CUN codon family was redefined to threonine, dueto the appearance of a mutant Thr-tRNA that containeda 5′-UAG-3′ anticodon that was able to decode thosefour codons using extended wobble rules. This mutantThr-tRNA was not aminoacylated by the standard ThrRS.Rather, it required a novel synthetase, which is not ableto aminoacylate the standard yeast Thr-tRNAs. This isthe only known case where an aminoacyl-tRNA synthetaseplays a direct role in a genetic code change. As in thecase of the Candida CUG codon, CUN reassignment wascomplete upon the disappearance of the Leu-tRNAUAGthat decoded these codons as leucine. (C) UGA identityredefinition in yeast mitochondria. The identity of the UGAnonsense codon to Trp in yeast mitochondria (and otherorganisms) was redefined gradually through ambiguousdecoding, involving an intermediary stage characterized bycompetition between nonsense suppressor tRNAs and therelease factor complex. The appearance of a Trp-tRNAsuppressor tRNA able to decode the UGA stop codon setthe stage for the identity redefinition, since it competedwith the mitochondrial release factor-2 (mRF2). Completereassignment of the UGA codon required a mutation in themRF2 gene to prevent it from recognizing the stop codon

Gln Arg Lys Ser

CognateLeu - tRNA

Leu Ser MutantSer - tRNA

Gln Arg Lys Ser

Ser MutantSer-tRNA

LeuOnly some Ser-codonsmutate to CUG

“old”

“new“

tRNAselection

5' CAG CUG CGG AAA UCN 3'

5' CAG UUG CGG AAA CUG 3'

Figure 4. Misreading of the CUG codon by a mutantSer-tRNA reduces its usage. Like biased genome G + Cpressure, codon misreading has a strong negative impacton codon usage. In the case of the Candida CUG codon,the appearance of the mutant Ser-tRNACAG introduced amisreading event that drove most of the 26 000–30 000CUG codons existent in the yeast ancestor almost toextinction. Only 2% of those codons remain in the C. albicansORFeome. The vanished codons mutated to leucine UUGor UUA codons and only very few mutated to the otherleucine codons (CUU, CUA and CUC). Interestingly, thepresence of the mutant Ser-tRNACAG created a selectiveforce that drove the migration of serine (UCN) codons toCUG, since many of the CUG codons present in the CandidaORFeome are represented by serine codons or codons ofamino acids conserved of serine in the ORFeomes of S.cerevisiae, C. glabrata and Sz. pombe. This explains why the17 000 CUG codons present in the C. albicans ORFeome arenot related to the CUG codons present in the ORFeomesof those yeasts, and shows that genetic code changes are adynamic process involving loss and gain of the codons beingreassigned [12]

allow it to become ambiguous, as such ambigu-ity affects a small number of proteins. Further-more, recent comparative genomics studies aimedat elucidating the pathway of CUG reassignmentin C. albicans have shown that codon ambigu-ity itself lowers codon usage and may even forceambiguous codons to disappear from ORFeomes[12] (Figure 4). Therefore, codon misreading andbiased G + C pressure have identical and syner-gistic effects on codon usage, but the former maytrigger a codon identity change directly through amisreading tRNA, while G + C pressure decreasescodon usage only [20].

Evolution of genetic code alterations inyeast

The reassignments of the four leucine CUN codonsto threonine in the mitochondria of S. cerevisiae

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 203–213.

Yeasts genetic code 207

and C. glabrata and of the leucine CUG codonto serine in Candida and Debaryomyces are notrelated to G + C biases. Instead, they are medi-ated through structural alteration of the trans-lational machinery, are driven by selection andstrongly support the ‘ambiguous intermediate the-ory’ [25,30]. For these reasons, they are excel-lent models for testing the impact of selection-driven genetic code alterations on physiology, geneexpression and the evolution of new species.

Reassignment of the leucine CUN codon tothreonine in yeast mitochondria

The reassignment of the yeast mitochondrialleucine CUN codon family to threonine is aclear case of a genetic code change mediated bystructural alteration of the translational machineryinvolving both tRNAs and aminoacyl-tRNA syn-thetases [17]. In S. cerevisiae mitochondria, anunusual threonine tRNA (thr-tRNAUAG) decodesthe four CUN codons (N = any nucleotide). ThistRNA has six base pairs in the anticodon steminstead of the canonical five base pairs, and sixnucleotides in the anticodon loop instead of thecanonical seven (Figure 5A). In C. glabrata, thehomologous Thr-tRNAUAG, which also decodes thefour CUN leucine codons as threonine, has sevencanonical nucleotides in its anticodon loop but,like the S. cerevisiae thr-tRNAUAG, contains sixbase pairs in the anticodon stem instead of thecanonical 5 base pairs (Figure 5A). Also, the sixthbase pair is a non-Watson–Crick U–U base pair,indicating that the top of the stem is flexible ormay even be open. In contrast, the yeast cyto-plasmic Thr-tRNAUGU, which decodes the standardThr ACN codon family has a standard anticodonstem (5 bp) and anticodon loop (7 bp), indicatingthat these structural alterations of the mitochondrialThr-tRNAUAG are linked to the change of iden-tity of the CUN codons from leucine to threonine[14] (Figure 5B). Furthermore, this reassignmentalso required a novel Thr-tRNA synthetase (ThrRS)that specifically aminoacylates the unique mito-chondrial Thr-tRNAUAG [11], indicating that CUNreassignment evolved gradually through structuralchange of both the Thr-tRNA and its cognateThrRS.

Another alteration of the translational machineryinvolves the reassignment of the UGA stop codonto tryptophan in the mitochondria of yeasts and

MitochondrialtRNATrp

S. cerevisiae

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AA

C

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A

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A - U

U - AG - C

U - AU - AC - GG - C

G - UU - AU - AG - C

N U N U

U . U

Figure 5. Structural novelties of the yeast mitochondrialtRNAs involved in genetic code alterations. (A) In S.cerevisiae and C. glabrata mitochondria, the leucine CUNcodon family is reassigned to threonine by novel Thr-tRNAsthat have six base pairs in the anticodon stem insteadof the canonical five base pairs. Interestingly, the firstbase pair of the stem is a non-Watson–Crick base pair(U–U), suggesting that these tRNAs have an atypicalanticodon arm. Furthermore, the S. cerevisiae Thr-tRNAonly has six nucleotides in the anticodon loop insteadof the canonical seven nucleotides, suggesting that eitherthe first base pair (U–U) of the stem does not form(creating a eight-nucleotide loop) or that the loop of thistRNA works with six nucleotides only. How these uniquestructural alterations influence decoding accuracy is not yetunderstood. However, eight-nucleotide loops are knownto induce frameshifting, and consequently it is likely thatthese unusual tRNAs have novel decoding properties. ThesetRNAs are also aminoacylated by a unique ThrRS that doesnot recognize the canonical Thr-tRNAs. (B) Conversely,the cytoplasmic Thr-tRNAUGU, which decodes the thrACA and ACG codons in the standard fashion, hasa standard anticodon arm (with the exception of thenon-Watson–Crick base pair at the bottom of the stem).(C) The other mitochondrial tRNA that mediates a geneticcode change in yeasts is the Trp-tRNAACA. In the standardgenetic code, the UGG codon is decoded by a singleTrp-tRNA with a 5′-CCA-3′ anticodon. However, in caseswhere both UGG and UGA are decoded as Trp theanticodon of that tRNA mutated to 5′-U*CA-3′. In this case,the uridine at the first anticodon position (U*) is modifiedto prevent decoding of the cysteine UGC and UGU codonsas tryptophan. As described in the text, the reassignment ofthe UGA codon to Trp required the disappearance of themRF2 that recognized this codon and terminated mRNAtranslation

other eukaryotes [7]. In this case, the release fac-tor that recognizes the UGA codon (mRF2) lostthis ability, thus setting the stage for capture ofthe UGA stop by a near-cognate tryptophan tRNA

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208 I. Miranda, R. Silva and M. A. S. Santos

(Figure 5C). In prokaryotes (and, most likely, inprimordial mitochondria), translation of the threestop codons requires two release factors, RF1 todecode UAG and UAA and RF2 to decode UAAand UGA. The capture of UGA by the Trp-tRNAcreated a new situation in which the mitochon-drial RF1 (mRF1) was sufficient to recognize thetwo remaining stop codons (UAG and UAA). Inother words, the requirement of a release factorfor decoding the UGA codon disappeared and con-sequently such a factor was eliminated from themitochondrial genome (Figure 3C). As before, thecapture of the UGA stop codon by the primor-dial Trp-tRNA is likely to have been mediatedby codon misreading, since a number of wild-typeTrp-tRNAs from various organisms suppress UGAnonsense mutations [8]. However, efficient decod-ing of the UGA as Trp required additional muta-tions in the anticodon of the primordial wild-typeTrp-tRNA (5′-CmCA-3′ anticodon), e.g. a C to Umutation in the first position of the anticodon tocreate the 5′-UCA-3′ anticodon (Figure 5C).

Reassignment of the CUG codon in thecytoplasm of Candida and Debaryomyces

The reassignment of the CUG codon from leucineto serine in the cytoplasm of Candida spp. andDebaryomyces spp. is also mediated through struc-tural alteration of the translation machinery [18,21].In these cases, an insertion of one adenosine in theintron of a Ser-tRNACGA (decodes the UCG ser-ine codon) generated a 5′-CAG-3′ anticodon andcreated a novel tRNA (Ser-tRNACAG

leu) that coulddecode the leucine CUG codon as serine [13,28].This created a new situation in which the CUGcodon was decoded by two tRNAs belonging totwo different amino acid families, i.e. a standardLeu-tRNA plus the newly generated Ser-tRNACAG.In other words, the CUG codon became ambiguous,since either leucine or serine could be inserted inresponse to it during ribosome decoding and, forreasons not yet fully understood, such ambiguitywas not eliminated by natural selection.

Considering that the anticodon stem and loopof tRNAs evolve in a coordinated fashion toprovide the correct three-dimensional environ-ment for the stacking of the anticodon-bases,which modulates efficient codon-anticodon inter-action, the emergence of the 5′-CAG-3′ anticodonin the body of a Ser-tRNA was problematic.

C AGC A G

AInsertion

C A Gm1G37

SER SER

AA37A37

U33U33U33

Decodingaccuracyrecovered

C A GG33

Latemutation

Step3Step2Step1

High LeuSER/Leu

Low LeuSER/Leu

Inaccuratedecoder

m1G37Loop Intron IntronLoop Loop Intron Loop

Intron

Displacedinto intron

Figure 6. Evolution of the Candida Ser-tRNACAG. TheSer-tRNACAG was created by an insertion of an adenosinein the middle position of the anticodon of a Ser-tRNACGAwhich decodes the UCG serine codon. This serine tRNAhas 71% identity with the C. albicans Ser-tRNACAG andhas an intron located 3′ to the anticodon. The adenosineinsertion displaced the splice-site by one nucleotide in the3′ direction and transformed the 5′-CGA-3′ anticodon intothe 5′-CAG-3′ anticodon, thus creating a serine tRNA thatcould decode the leucine CUG codon as serine [12]. Theresulting Ser-tRNACAG had adenosine in position 37 (A37),as is typical of Ser-tRNAs. However, leucine 5′-CAG-3′anticodons use m1G37 to prevent frameshifting. Therefore,the late introduction of G37 (step2) resulted from theimperative to maintain decoding accuracy but also permittedrecognition of the tRNA by the LeuRS, since this synthetaserecognizes both the methyl group of G37 and the adenosinein the middle position of the anticodon (A35). This createda new situation in which the Ser-tRNACAG was charged byboth the LeuRS and the SerRS, which would have preventedfull reassignment of the CUG codon from leucine to serine.In the last stage (stage 3) of the evolutionary pathway,U33 mutated to G33 and lowered the leucylation efficiencyof the Ser-tRNACAG, since it distorted the anticodon armand lowered binding efficiency of the LeuRS to the tRNA.That is, while G37 is imperative for decoding accuracy, G33lowered CUG ambiguity to the 3–5% level

The context of the 5′-CAG-3′ anticodon in allorganisms is 5′-U33-C34-A35-G36-m1G37-3′. How-ever, the context of the 5′-CAG-3′ anticodon inthe primordial Ser-tRNACAG would have been5′-U33-C34-A35-G36-A37-3′, since serine tRNAshave A at position N37. The 5′-CAG-3′ anticodonshave m1G37 and lack of methylation of G37 inducesframeshifting [3], suggesting that the mutant Ser-tRNACAG containing A37 was an inaccurate andinefficient decoder (Figure 6). This explains whythe Ser-tRNACAG encoded by several Candidaspecies shows greater than expected anticodon-arm variability and, with the exception of the Ser-tRNACAG from Candida cylindracea, have m1G37[29]). In other words, the presence of a leucine5′-CAG-3′ anticodon in the body of a serine tRNAcreated a selective pressure for additional structural

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 203–213.

Yeasts genetic code 209

change that restored decoding accuracy to standardlevels (Figure 6).

Remarkably, such a decoding imperative createdan unique aminoacylation problem, due to insertionof two major identity elements for the leucyl-tRNAsynthetase (LeuRS) in the anticodon loop of themutant Ser-tRNACAG (5′-G33-C34-A35-G36-m1G37-3′), namely A35 and the methyl group of G37(Figure 7), which allowed for its recognition bythis enzyme [26]. Conversely, the seryl-tRNA syn-thetase (SerRS) recognizes the run of three G–Cbase pairs of the extra loop plus the discriminatorbase of Ser-tRNAs [4], which were not altered inthe Ser-tRNACAG and, consequently, the tRNACAGwas also charged with serine. In conclusion, A35and m1G37 created a hybrid tRNA that could becharged by both LeuRS and SerRS [27] (Figure 7)and reintroduced the ambiguity of the CUG codonthat was initially created through competition of theLeu-tRNACAG with the mutant Ser-tRNACAG (seebelow; Figure 9).

This ambiguity of the CUG codon created bym1G37 was lowered at a later stage by an additionalmutation in the anticodon loop of the Ser-tRNACAGthat replaced the highly conserved U33 with G33(Figures 6 and 7). The U33 ensures the correct

turn (U-turn) of the phosphate backbone in theanticodon loop of tRNAs which is critical forthe correct stacking of the anticodon bases. Inthe case of the Ser-tRNACAG, G33 distorts theRNA helix of the anticodon stem and preventsefficient recognition of the Ser-tRNACAG by theLeuRS [18]. In doing so, G33 allowed for CUGreassignment to proceed to near completion in mostCandida species (see Figures 6, 7). Interestingly,in Candida cylindracea the CUG codon has beenfully reassigned to Ser due to the maintenance ofA37 in its Ser-tRNACAG (Figure 8). It is not yetclear why CUG ambiguity was maintained in somespecies and was eliminated in others, and howthe C. cylindracea Ser-tRNACAG overcame thenecessity of having m1G37 for accurate decoding.

Time scale of CUG reassignment

In order to gain new insight on the evolutionarypathway of CUG reassignment, our group usedmolecular phylogeny analysis, based on rRNAand tRNA sequences, to time the critical stepsof CUG identity redefinition [12]. These studiesshowed that high-level serine-CUG decoding is

A

SERINE 95%Leucine ± 5%

G CA UU GA UC GG CA U

C G U C CG C A G G

C m1 A1

G

ψψψψT

A

C

C

m5C5CG

A

G

m 2 G2

D U

C

CC

Gm1G

GG

G

ψψψψU

U

U

A

A UA UG CG CA ψψψψψψψψ

G33

U

C C GG G C

A

A

Gm

GG

D D

G

ACCG73 Discriminator

2

ac44

CG

Purine

Pyrimidine U33

Nucleotide 33

Interactionwith SerRS

Nucleotide 73(discriminator base)

Interactionwith LeuRS

Distortioninduced by G33

Figure 7. Secondary and tertiary structures of the C. albicans Ser-tRNACAG, which are hybrid molecules that have a bodyof a serine tRNA and the anticodon arm of leucine tRNAs. They contain identity elements (nucleotides that are recognizedby the aaRSs) for both the SerRS, namely the three G–C base pairs of the extra arm (red) and the discriminator base (G73),and for the LeuRS, viz. A35 and m1G37 in the anticodon loop, as shown in the three-dimensional model of Ser-tRNACAG tothe right of the figure. The distortion indicated in the three-dimensional model has been determined in vitro in solution,using chemical and enzymatic probing of the structure. Replacement of G33 with U33 increases leucylation efficiency of theSer-tRNACAG in vitro, showing that the main function of G33 is to keep CUG ambiguity at low level [18]

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210 I. Miranda, R. Silva and M. A. S. Santos

A - UG - CG - C

A27- U

A - ΨΨΨΨU

G

C A G

m1GΨΨΨΨ

28 42

29 41

30 40

43

31 39393939

32

331

37

38383838 G - CG - CC - G40

- A

G ΨΨΨΨU

CC A G

m1GΨΨΨΨ

ΨΨΨΨ28 42

29 41

30

27 43

31 - 39393939

32

37

33338888

U28 - A42

G29- C41

G30 - C40

27 U43

A31 - ΨΨΨΨ39C32

G33

C G

ΑΑΑΑ38

A

A

ΨΨΨΨ

C. cylindracea C. tropicalis

C. albicansC. parapsilosisC. zeylanoidesC. maltosaC. melibiosica

100% Ser± 97% Ser

1 - 7% Leu?

-

Figure 8. Evolutionary pathways of Ser-tRNACAG in Candida spp. The anticodon arm of the Ser-tRNACAG from variousCandida spp. shows three different evolutionary ‘strategies’ for CUG reassignment. In the case of C. cylindracea, thereassignment to serine has been fully accomplished, since this Ser-tRNACAG has A37, which prevents recognition of thetRNA by the LeuRS. In most Candida species, the Ser-tRNACAG contains G37, whose methyl group is recognized by theLeuRS, thus allowing for charging of the tRNA with leucine (up to 7%) and creating CUG ambiguity, since the same tRNAis also charged with serine by the SerRS (up to 93%). On the other hand, in C. tropicalis, the canonical U33 was replaced byanother pyrimidine (C) and not by a purine (G). In contrast to G33, the C33 mutation does not distort the anticodon armof the Ser-tRNACAG, and consequently this tRNA should be charged at higher levels with leucine. However, the level ofambiguity of the C. tropicalis CUG codon has not yet been determined in vivo

approximately 171 ± 27 million years old; how-ever the Ser-tRNACAG originated at least 272 ±25 million years ago. Similar molecular phylogenyanalysis indicated that the genera Candida andSaccharomyces separated from each other 178 ±19 million years ago, implying that the CUGcodon was highly ambiguous for approximately100 million years in the ancestor of yeasts [12](Figure 9). The exception to this is the genusSchizosaccharomyces, which separated much ear-lier from the other yeasts (Figure 9).

The maintenance of CUG decoding ambiguityover an evolutionary time scale prompts the fol-lowing fundamental biological questions: (a) howhas such ambiguity affected codon usage?; and(b) what was the impact of such ambiguity on theevolution of yeasts? The answer to the first ques-tion was obtained through a comparative genomicsapproach, using a set of orthologous genes fromC. albicans, S. cerevisiae and Schizosaccharomycespombe and identifying the positions occupied bythose CUG codons in these ORFeomes. Remark-ably, CUG ambiguity forced the disappearance ofapproximately 26 000–30 000 CUG codons thatexisted in the Candida ancestor and strongly

Ancestor

C. albicansCUG = Ser + Leu

S. cerevisiaeCUG = Leu

272 ± 25million years

170 ± 10million years

Sz. pombeCUG = Leu

420 MY

Leu Ser

CUG

C. cylindraceaCUG = Ser

Figure 9. The evolutionary pathway of CUG reassign-ment in Candida. rRNA and tRNA molecular phylogenyindicates that the Ser-tRNACAG appeared approximately272 ± 25 million years ago. This mutant Ser-tRNA com-peted with the standard Leu-tRNA for the CUG codon,introducing ambiguity at the CUG codon during mRNAdecoding. Interestingly, the genera Candida and Saccha-romyces separated from each other 170 million years ago,indicating that the yeast ancestor was ambiguous for atleast 100 million years. The genus Schizosaccharomyces wasnot affected by such CUG ambiguity, since it separatedfrom the other yeast genera prior to the appearance ofthe Ser-tRNACAG. The Ser-tRNACAG was maintained in thelineage that originated the genus Candida, but was lost inthe lineage leading to the genus Saccharomyces. In the genusCandida, some species still retain CUG ambiguity, whileothers have achieved complete reassignment of the CUGcodon [12]

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 203–213.

Yeasts genetic code 211

influenced the usage of the other codons belong-ing to the CUN codon family. Only 2% of theseC. albicans CUG codons are represented by oneof the other five leucine codons (CUA, CUC,CUU, UUG and UUA) in orthologous genes ofS. cerevisiae and Sz. pombe. The remaining 98%of CUG codons appear in positions representedby serine codons or codons specifying conservedamino acids of serine [12]. That is, the 30 000 and26 000 CUG codons present in the S. cerevisiaeand Sz. pombe ORFeomes, respectively, are notrelated to the 17 000 CUG codons present in theORFeome of extant C. albicans, since the lattercodons evolved during the last 272 ± 25 millionyears from codons denoting serine rather thanleucine. This implies that CUG identity redefinitionwas mediated through a dynamic mechanism thatinvolved gradual disappearance of ‘old’ ambiguousLeu-CUG codons (coding for leucine + serine) andsimultaneous emergence of ‘new’ ambiguous Ser-CUG codons arising via mutation of codons thatencoded serine or conserved amino acids of serine[12] (Figure 4).

This novel pathway of codon identity redefinitionclearly shows that C. albicans, and most likely allother organisms, are well equipped to tolerate thenegative impact of proteome disruption caused bycodon identity redefinition. Additionally, it impliesthat the C. albicans proteome has been unsta-ble during the last 272 ± 25 million years, sincethe disappearing (old) and emerging (new) codonsremained ambiguous from the initial appearance ofthe Ser-tRNACAG to the present day [27]. Further-more, the appearance of the novel Ser-tRNACAGwas a major evolutionary force shaping the usageof all six leucine codons (UUA, UUG, CUA, CUG,CUC and CUU), showing that redefinition of theidentity of a single codon affects the usage of allcodons belonging to a codon family [12].

Other implications of CUG reassignment

The studies described above indicate that the pro-teome of yeasts was unstable on a geological timescale and suggest that such ambiguity may some-how be advantageous. The fact that it was elimi-nated in C. cylindracea through structural alterationof the anticodon loop of the Ser-tRNACAG pro-vides clear evidence that it does not represent anevolutionary or structural death ending created by

the imperative of maintaining decoding accuracy.Therefore, one is prompted to ask:

1. How did the ancestor of yeasts dealt with suchproteome destabilization?

2. What are the consequences of proteome destabi-lization on physiology, gene expression, geneticvariability and adaptation?

3. Are ambiguous Candida and Debaryomycesquasi-species?

Considering that approximately 272 million yearshave passed since the appearance of the Ser-tRNACAG, it is impossible to answer these ques-tions directly. However, it is still possible to recon-struct the evolutionary pathway of CUG reassign-ment in S. cerevisiae in order to gain some insightinto these problems. Moreover, genetic code ambi-guity can be viewed in a wider context of gen-eral mRNA mistranslation, and it is likely that itsstudy will provide important new insights on howthe genetic code evolves and also on the conse-quences of mRNA mistranslation for cell evolutionand degeneration. Recent studies showed that trans-formation of S. cerevisiae cells with the C. albicansSer-tRNACAG mimics the natural ambiguity of thelatter species, since the CUG codon is decoded asboth leucine and serine. These ambiguous cells arebeing used to study the impact of codon identityredefinition on physiology and adaptation, usingglobal genomics approaches, i.e. phenotypic arrays,DNA-microarrays and proteomics [20]. Ongoingstudies in our laboratory validate this experimentalapproach, as ambiguous S. cerevisiae cells displaynew phenotypes, such as high tolerance to heavymetals, drugs, ethanol, oxidants and sodium chlo-ride [22]. Although these cells have a growth dis-advantage in rich media, they display significantselective advantages under extreme environmentalstress conditions. The molecular mechanism thatmediates such tolerance to stress is not yet under-stood, but it is likely that the synthesis of aberrantproteins caused by CUG ambiguity triggers expres-sion of molecular chaperones (Hsp104 and Hsp70),that protect ambiguous cells from severe stress.Therefore, the redefinition of the identity of theCUG codon over 272 ± 25 million years is likelyto have had a major impact on the stress responseand evolution of new traits in the genera Candidaand Debaryomyces. This principle should apply toall organisms that have evolved genetic code alter-ations.

Copyright 2006 John Wiley & Sons, Ltd. Yeast 2006; 23: 203–213.

212 I. Miranda, R. Silva and M. A. S. Santos

Considering that the Candida ancestor had26 000–30 000 CUG codons, and that extant C.albicans still has 17 000 CUG codons distributedover 50% of its ORFeome at a frequency of 1–10CUG/ORF [12], CUG decoding as both serineand leucine implies that mRNA containing CUGcodons originate arrays of different proteins. Inother words, C. albicans proteins (and other Can-dida and Debaryomyces proteins) are not truechemical entities. The proteome therefore has astatistical nature, and for this reason these yeastsshould be considered quasi-species. The conceptof quasi-species is normally associated with virusesthat replicate their genomes with low fidelity, butsuch a concept also applies to ambiguously codingyeasts, since low fidelity in both DNA replicationand in mRNA translation results in a statisticalproteome. This may explain the high genetic insta-bility and the high heterozygosity of the C. albicansgenome because mutant DNA polymerases andrepair enzymes, which may replicate the genomewith high error rates, are permanently synthesized.That is, CUG-decoding ambiguity may result in ahypermutagenic phenotype similar to that observedin ambiguous E. coli cells [1,19].

Conclusions

The evolution of genetic code alterations in yeastsand in other lower eukaryotes, e.g. ciliates, pro-vides strong evidence for a critical role for mRNAmistranslation in the evolution of the genetic code.Mistranslation has been studied over a number ofyears, but its role in the evolution of the geneticcode has been largely overlooked. The study ofthe evolutionary pathway of CUG reassignmentin Candida and its reconstruction in S. cerevisiaehighlights how codon decoding ambiguity alterscodon usage, and how it provides a mechanism forcodon identity change. It is likely that most geneticcode alterations evolved through a codon misread-ing mechanism and required significant structuralalteration of the protein synthesis machinery. Thisis in line with the observation that expansion ofthe genetic code to selenocysteine and pyrrolysinerequired novel translational factors for reprogram-ming the UGA and UAG stop codons in bothbacteria and eukaryotes. The study of genetic codealterations and genetic code expansion is thereforeproviding a unique insight on how the genetic code

may have evolved from a minimal set of aminoacids to the 22 amino acids used in extant organ-isms.

Genetic code ambiguity induces pleiotropic phe-notypic alterations that can only be tackled usinggenomics approaches. The availability of the com-plete genome sequences of various yeasts and theexistence of a large array of genetics, genomics,proteomics and systems biology tools for S. cere-visiae provides unique opportunities to dissect theevolution of CUG reassignment in detail, thusputting this yeast at the forefront of research intothe evolution of the genetic code.

AcknowledgementsResearch in the Santos laboratory is supported by FCT/FEDER project grant REF: POCI/BIA-MIC/55466/04 andREEQ/737/2001. I.M. is supported by FCT/FEDER PhD,BD/19807/99; R.S. is supported by a FCT grant SFRH/BPD/20683/2004; M.A.S.S. is an EMBO YIP and hiswork is supported by the FCT, through grants from thePOCI/FEDER programme and by the Human FrontierScience Programme (Grant RGP45/2005).

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