How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

A unified model of codon reassignment in alternative genetic codes

Many modified genetic codes are found in specific genomes in which one or more codons have been reassigned to a different amino acid from that in the canonical code. We present a new framework for codon reassignment that incorporates two previously proposed mechanisms (codon disappearance and ambiguous intermediate) and introduces two further mechanisms (unassigned codon and compensatory change). Our theory is based on the observation that reassignment involves a gain and a loss. The loss could be the deletion or loss of function of a tRNA or release factor. The gain could be the gain of a new type of tRNA or the gain of function of an existing tRNA due to mutation or base modification. The four mechanisms are distinguished by whether the codon disappears from the genome during the reassignment and by the order of the gain and loss events. We present simulations of the gain-loss model showing that all four mechanisms can occur within the same framework as the parameters are varied. We investigate the way the frequencies of the mechanisms are influenced by selection strengths, the number of codons undergoing reassignment, directional mutation pressure, and selection for reduced genome size.     

Tandem Stop Codons in Ciliates That Reassign Stop Codons

Tandem stop codons are extra stop codons hypothesized to be present downstream of genes to act as a backup in case of read-through of the real stop codon. Although seemingly absent from Escherichia coli, recent studies have confirmed the presence of such codons in yeast. In this paper we will analyze the genomes of two ciliate species—Paramecium tetraurelia and Tetrahymena thermophila—that reassign the stop codons TAA and TAG to glutamine, for the presence of tandem stop codons. We show that there are more tandem stop codons downstream of both Paramecium and Tetrahymena genes than expected by chance given the base composition of the downstream regions. This excess of tandem stop codons is larger in Tetrahymena and Paramecium than in yeast. We propose that this might be caused by a higher frequency of stop codon read-through in these species than in yeast, possibly because of a leaky termination machinery resulting from stop codon reassignment.     

Development of the genetic code: Insights from a fungal codon reassignment

The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution.     

Reassignment of sense codons in vivo

The genetic code maps one or more of the 61 sense codons onto a set of 20 canonical amino acids. Reassignment of sense codons to non-canonical amino acids in model organisms such as Escherichia coli has been achieved through manipulation of the cellular protein synthesis machinery. Specifically, control of amino acid pools, coupled with engineering of the aminoacyl-tRNA synthetase activity of the host, has enabled assignment of sense codons to a wide variety of non-canonical amino acids under conditions routinely used for expression of recombinant proteins. Codon reassignment is leading to important advances in protein engineering and bioorganic chemistry. Here we summarize some of those advances, and provide detailed protocols for codon reassignment.     

UAA and UAG may Encode Amino Acid in Cathepsin B Gene of Euplotes octocarinatus

The ciliate Euplotes deviates from the universal genetic code by translating UGA as cysteine and using UAA and UAG as the termination codon. Here, we cloned and sequenced the Cathepsin B gene of Euplotes octocarinatus (Eo‐CTSB) which containing several in‐frame stop codons throughout the coding sequence. We provide evidences, based on 3′‐RACE method and Western blot, that the Eo‐CTSB gene is actively expressed. Comparison of the derived amino acid sequence with the homologs in other eukaryotes revealed that UAA and UAG may code for glutamine in Eo‐CTSB. These findings imply an evolutionary complexity of stop codon reassignment in eukaryotes.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure-function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2-7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

How Mitochondria Redefine the Code

Annotated, complete DNA sequences are available for 213 mitochondrial genomes from 132 species. These provide an extensive sample of evolutionary adjustment of codon usage and meaning spanning the history of this organelle. Because most known coding changes are mitochondrial, such data bear on the general mechanism of codon reassignment. Coding changes have been attributed variously to loss of codons due to changes in directional mutation affecting the genome GC content (Osawa and Jukes 1988), to pressure to reduce the number of mitochondrial tRNAs to minimize the genome size (Anderson and Kurland 1991), and to the existence of transitional coding mechanisms in which translation is ambiguous (Schultz and Yarus 1994a). We find that a succession of such steps explains existing reassignments well. In particular, (1) Genomic variation in the prevalence of a codon's third-position nucleotide predicts relative mitochondrial codon usage well, though GC content does not. This is because A and T, and G and C, are uncorrelated in mitochondrial genomes. (2) Codons predicted to reach zero usage (disappear) do so more often than expected by chance, and codons that do disappear are disproportionately likely to be reassigned. However, codons predicted to disappear are not significantly more likely to be reassigned. Therefore, low codon frequencies can be related to codon reassignment, but appear to be neither necessary nor sufficient for reassignment. (3) Changes in the genetic code are not more likely to accompany smaller numbers of tRNA genes and are not more frequent in smaller genomes. Thus, mitochondrial codons are not reassigned during demonstrable selection for decreased genome size. Instead, the data suggest that both codon disappearance and codon reassignment depend on at least one other event. This mitochondrial event (leading to reassignment) occurs more frequently when a codon has disappeared, and produces only a small subset of possible reassignments. We suggest that coding ambiguity, the extension of a tRNA's decoding capacity beyond its original set of codons, is the second event. Ambiguity can act alone but often acts in concert with codon disappearance, which promotes codon reassignment. Received: 26 October 2000 / Accepted: 19 January 2001     

Ciliates use both variant and universal genetic codes: evidence of omnipotent eRF1s in the class Litostomatea

Stop codon reassignments have occurred very frequently in ciliates. In some ciliate species, the universal stop codons UAA and UAG are translated into glutamine, while in some other species, the universal stop codon UGA appears to be translated into cysteine or tryptophan. The class Litostomatea has been hypothesized to be the only group of ciliates using the universal genetic code. However, the hypothesis was based on a statistical analysis of quite small sequence dataset which was insufficient to elucidate the codon usage of the class among such highly deviated phylum. In this study, together with the updated database sequence analysis for the class, we approached the problem of stop codon usage by examining the capacity of the translation termination factor eRF1 for recognizing stop codons. Using in vivo assay systems in budding yeast, we estimated the activity of eRF1 from two litostome species Didinium nasutum and Dileptus margaritifer. The results clearly showed that Didinium and Dileptus eRF1s efficiently recognize all three stop codons. This is the first experimental evidence that strongly supports the hypothesis that litostome ciliates use universal genetic code.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure–function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2–7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

Release Factor One Is Nonessential in Escherichia coli

Recoding a stop codon to an amino acid may afford orthogonal genetic systems for biosynthesizing new protein and organism properties. Although reassignment of stop codons has been found in extant organisms, a model organism is lacking to investigate the reassignment process and to direct code evolution. Complete reassignment of a stop codon is precluded by release factors (RFs), which recognize stop codons to terminate translation. Here we discovered that RF1 could be unconditionally knocked out from various Escherichia coli stains, demonstrating that the reportedly essential RF1 is generally dispensable for the E. coli species. The apparent essentiality of RF1 was found to be caused by the inefficiency of a mutant RF2 in terminating all UAA stop codons; a wild type RF2 was sufficient for RF1 knockout. The RF1-knockout strains were autonomous and unambiguously reassigned UAG to encode natural or unnatural amino acids (Uaas) at multiple sites, affording a previously unavailable model for studying code evolution and a unique host for exploiting Uaas to evolve new biological functions.     

A thermodynamic model of protein structure evolution explains empirical amino acid substitution matrices

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. These evolutionary pressures are sufficiently consistent over time and across protein families to produce substitution patterns, summarized in global amino acid substitution matrices such as BLOSUM, JTT, WAG, and LG, which can be used to successfully detect homologs, infer phylogenies, and reconstruct ancestral sequences. Although the factors that govern the variation of amino acid substitution rates have received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid substitution matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi‐nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex yet universal pattern observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary driver behind the global amino acid substitution patterns observed in proteins throughout the tree of life.     

On the organizational dynamics of the genetic code

The organization of the canonical genetic code needs to be thoroughly illuminated. Here we reorder the four nucleotides-adenine, thymine, guanine and cytosine-according to their emergence in evolution, and apply the organizational rules to devising an algebraic representation for the canonical genetic code. Under a framework of the devised code, we quantify codon and amino acid usages from a large collection of 917 prokaryotic genome sequences, and associate the usages with its intrinsic structure and classification schemes as well as amino acid physicochemical properties. Our results show that the algebraic representation of the code is structurally equivalent to a content-centric organization of the code and that codon and amino acid usages under different classification schemes were correlated closely with GC content, implying a set of rules governing composition dynamics across a wide variety of prokaryotic genome sequences. These results also indicate that codons and amino acids are not randomly allocated in the code, where the six-fold degenerate codons and their amino acids have important balancing roles for error minimization. Therefore, the content-centric code is of great usefulness in deciphering its hitherto unknown regularities as well as the dynamics of nucleotide, codon, and amino acid compositions.     

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA^Pyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA^Pyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.     

Removing the redundancy from randomised gene libraries

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NN(G)(CorT) (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins.     

Mitochondrial Genetic Codes Evolve to Match Amino Acid Requirements of Proteins

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular—and contrary to the predictions of the mutation-drift hypothesis—the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.     

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.     

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.     

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.