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COMICR-698; NO OF PAGES 7 Available online at www.sciencedirect.com 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 www.sciencedirect.com 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 Current Opinion in Microbiology 2009, 12:1–7 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 COMICR-698; NO OF PAGES 7 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 www.sciencedirect.com 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 COMICR-698; NO OF PAGES 7 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. www.sciencedirect.com Current Opinion in Microbiology 2009, 12:1–7 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 COMICR-698; NO OF PAGES 7 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 www.sciencedirect.com 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 COMICR-698; NO OF PAGES 7 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]. 5. Parker J, Precup J: Mistranslation during phenylalanine starvation. Mol Gen Genet 1986, 204:70-74. 6. Precup J, Parker J: Missense misreading of asparagine codons as a function of codon identity and context. J Biol Chem 1987, 262:11351-11355. 7. Precup J, Ulrich AK, Roopnarine O, Parker J: Context specific misreading of phenylalanine codons. Mol Gen Genet 1989, 218:397-401. 8. Stansfield I, Jones KM, Kushnirov VV, Dagkesamanskaya AR, Poznyakovski AI, Paushkin SV, Nierras CR, Cox BS, TerAvanesyan MD, Tuite MF: The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J 1995, 14:4365-4373. 9. Song H, Mugnier P, Das AK, Webb HM, Evans DR, Tuite MF, Hemmings BA, Barford D: The crystal structure of human eukaryotic release factor eRF1 — mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 2000, 100:311-321. 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. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Ling J, Reynolds N, Ibba M: Aminoacyl-tRNA synthesis and translational quality control. Annu Rev Microbiol 2009, 63:61-78. Comprehensive review of the editing mechanisms of aminoacyl-tRNA synthetases. The authors also discuss the biological implications of translational error. 2. Ogle JM, Ramakrishnan V: Structural insights into translational fidelity. Annu Rev Biochem 2005, 74:129-177. 3. Kramer EB, Farabaugh PJ: The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA 2007, 13:87-96. The authors developed a highly sensitive luminescent methodology to study translational error in vivo in E. coli. They demonstrate that average decoding error in vivo is 3.4 104 per codon and that it varies widely between codons. A main source of error is the competition between tRNAs at the ribosome A-site. 4. Stahl G, Salem SN, Chen L, Zhao B, Farabaugh PJ: Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae. Eukaryot Cell 2004, 3:331-338. www.sciencedirect.com 10. Eaglestone SS, Cox BS, Tuite MF: Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism. EMBO J 1999, 18:1974-1981. 11. Allmang C, Wurth L, Krol A: The selenium to selenoprotein pathway in eukaryotes: more molecular partners than anticipated. Biochim Biophys Acta 2009 doi: 10.1016/ j.bbagen.2009.03.003. Review of the eukaryotic selenosome machinery. The authors review the selenocysteine (Sec) biosynthesis pathway, in particular the enzymes that convert serine into phosphoserine and selenocysteine, and also the translation factors that are involved in Sec insertion at UGA codons. 12. 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Genetic code ambiguity: an unexpected source of proteome innovation and phenotypic diversity, Curr Opin Microbiol (2009), doi:10.1016/ j.mib.2009.09.004 COMICR-698; NO OF PAGES 7 6 Growth and development: eukaryotes 14. Gursinsky T, Grobe D, Schierhorn A, Jager J, Andreesen JR, Sohling B: Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a Grampositive anaerobe in Escherichia coli. Appl Environ Microbiol 2008, 74:1385-1393. 15. Turanov AA, Lobanov AV, Fomenko DE, Morrison HG, Sogin ML, Klobutcher LA, Hatfield DL, Gladyshev VN: Genetic code supports targeted insertion of two amino acids by one codon. Science 2009, 323:259-261. This study demonstrates that cysteine (Cys) and selenocysteine (Sec) are inserted at UGA codons in the ciliate Euplotes crassus and that such codon ambiguity is necessary to produce fully functional proteins. There are specialized tRNAs for Cys and Sec insertion at UGAs. 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The authors highlight how incorporation of artificial amino acids into the genetic code can be used to produce proteins with novel properties. Current Opinion in Microbiology 2009, 12:1–7 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