Cug codes for which amino acid

In the unassigned codon models, one of the 64 codons becomes untranslatable because its tRNA has been lost from the genome, or at least lost its function 3. The codon can later be captured by another amino acid if a tRNA for that amino acid mutates so that it can read the unassigned codon. Translation of the free codon is therefore disturbed or abolished 6 , but the loss of the original tRNA may be partially compensated by wobble decoding of the free codon by other tRNAs, including ones for other amino acids Depending on the extent to which the free codon is translatable by alloacceptor tRNAs, the tRNA loss-driven model can be regarded as a variant of the unassigned codon model if there is no translation or of the ambiguous intermediate model if alloacceptor tRNAs could read the codon by wobble, even before the original tRNA was lost.

Previous studies, both by experimentation and by comparative genomics, have shown that mutations in the anticodon of tRNA genes occur frequently 17 , These anticodon shifts can be synonymous, altering the balance between isoacceptor tRNAs for the same amino acid, or nonsynonymous, redeploying the tRNA to a codon for a different amino acid and so causing mistranslation Using whole-genome data to establish phylogeny, and mass spectrometry to determine genetic codes, we show that the CUG codon was reassigned on three separate occasions during the evolution of budding yeasts.

We discuss the mechanism of genetic code change, and the cause of the evolutionary instability of CUG-Leu translation in budding yeasts. To identify species with modified genetic codes, we systematically examined the genomes of 52 yeast species including 7 newly sequenced and two outgroups.

The species phylogeny was inferred by maximum likelihood from whole-genome amino acid data under a site-homogeneous model Fig.

An almost identical tree containing the same set of five clades was obtained using a site-heterogeneous model Supplementary Fig.

Phylogenomic tree and CUG decoding in 52 yeast species. Point X indicates the last common ancestor of the clades with altered genetic codes. Circles indicate the presence of tRNA genes with the indicated anticodons. Asterisks beside species names indicate genomes sequenced in this study. The tree was constructed from proteins by maximum likelihood. The data analysis pipeline is summarized in Supplementary Fig. The mass error values of CUG-translated residues were similar to those of other residues Supplementary Fig.

Examining the BLAST and mass spectrometry results in the context of the inferred species phylogeny, we conclude that there are five monophyletic groups clades that differ in their translations of CUG, which we refer to as the Ala, Ser1, Ser2, Leu1, and Leu2 clades Fig. The Ala clade contains the genera Nakazawaea and Peterozyma , as well as Pachysolen 11 , The split between the Ala and Leu2 clades forms a deep division within the yeast family Pichiaceae The Leu2 clade includes Citeromyces and Kuraishia , as well as the industrial yeasts Komagataella , Ogataea , and Pichia.

The newly identified Ser2 clade contains only the genera Ascoidea and Saccharomycopsis. Ser2 clade species have few CUG codons in conserved genes and gave conflicting results in the BLAST analysis, but the mass spectrometry data showed that CUG is translated as serine in the two species analyzed, Saccharomycopsis capsularis and Ascoidea rubescens Fig.

The Ser2 clade is sister to the Leu1 clade, which contains Saccharomyces cerevisiae and extends as deep as Cyberlindnera and Wickerhamomyces families Saccharomycetaceae, Saccharomycodaceae, and Phaffomycetaceae The branches separating the clades with different codes are short, so we evaluated the support for alternative topologies using the Shimodaira-Hasegawa and Approximately Unbiased bootstrapping tests Supplementary Table 2.

These analyses rejected the possibility that the Ser1 and Ser2 clades shared a common ancestor after they diverged from the Ala, Leu1, and Leu2 clades. They also rejected the hypothesis that the Leu1 and Leu2 clades are sisters. Therefore, the most parsimonious explanation of the data is that the CUG codon has been reassigned three times, on three separate branches of the Saccharomycotina tree: once from Leu to Ala, and twice from Leu to Ser Fig.

The tS CAG gene i. Specifically, the novel tS CAG genes in the two clades are derived from source genes that read the two different serine codon boxes Fig. To our knowledge, there is no precedent for a genome that naturally produces competing tRNAs that read the same codon but insert different amino acids. What made the CUG codon so unstable in yeasts? The meaning of sense codons in nuclear genes has been stable throughout all of eukaryotic evolution 2 , 3 , 4 , 5 , 6 , except in the three yeast clades where CUG became reassigned.

The observations that three reassignments occurred independently in three closely related eukaryotic lineages, and that they all involved the same codon, strongly suggest that the reassignments shared a common evolutionary cause. As described below, we hypothesize that the shared cause was natural selection acting against the ancestral tRNA Leu CAG , and that the selective pressure was caused by a killer toxin that attacked this specific tRNA molecule see Discussion.

These features are summarized here and described in more detail in Supplementary Note 3. First, the tL CAG gene was modified in some Leu1 clade species by unprecedented expansion of its intron, making it the largest canonical tRNA intron known — nt; Supplementary Fig.

The intron contains extensive secondary structure, which is likely to slow the rate of formation of the mature spliced and base-modified tRNA Leu CAG. Second, the gene was replaced in other Leu1 and Leu2 species represented by Pichia and Lachancea thermotolerans in Fig. Leu1 clade species also show other unusual deviations from the normal wobble rules used by eukaryotes, in the way they read the CUN codon box Supplementary Note 3.

Plus and minus symbols indicate inferred gains and losses of tRNA types. For each branch, only some representative genera or species are named. The coexistence of ancestral tL CAG and novel tS CAG genes in Ser2 clade species provides direct support for the ambiguous intermediate model of genetic code evolution 3 , The Ser2 clade species appear to be in the final stages of the evolutionary transition between genetic codes. The evolutionary conservation of tL CAG in four Ser2 clade species is puzzling, but a possible explanation is that tRNA Leu CAG may be required for translation of some genes specifically expressed in conditions that we did not examine, for example meiosis.

It suggests that some CUG sites in these four species still code for essential leucine residues. We propose that the genetic code changes were driven by natural selection against the existence of a particular tRNA species the ancestral form of tRNA Leu CAG , and not by selection in favor of the proteomic consequences of the code changes. Selection in favor of the proteomic changes seems implausible in view of the phylogenomic evidence that three independent reassignments occurred Fig.

In contrast, AlaRS and SerRS are the only two aminoacyl tRNA synthetases that do not require particular bases to be present in the anticodon of the tRNAs they charge 24 , so these are the only two amino acids to which CUG could have readily been reassigned from leucine simply by mutating the anticodon of an existing tRNA Ala or tRNA Ser 16 , although multiple mutations are required in the anticodon.

Our hypothesis of selection against tRNA Leu CAG is also consistent with the tendency for CUG codons to be located at non-essential sites in species that have changed their genetic code 9. CUG codons are relatively rare in the clades that changed codes. They tend to occur in orphan genes, or in regions of genes that do not align well with other species, as opposed to sites that are well conserved Supplementary Figs.

This pattern would not be expected if the genetic code changes were favored because of their effect on protein sequences. VLEs are cytoplasmic linear DNA plasmids also called killer plasmids that code for a toxin and an antitoxin 25 , The toxins are ribonucleases that cleave the anticodon loops of specific tRNAs. Similar, but uncharacterized, VLE-like plasmids have been described in Leu2 and Ser2 clade species 28 , In contrast, VLE-like sequences are absent from the genomes of the Leu0 species, an outcome whose probability is 0.

Some yeast lineages responded by changing their genetic codes, whereas others altered the sets of tRNA Leu genes they contain and managed to retain the standard code, either by changing their wobble rules or by acquiring versions of tRNA Leu CAG that were resistant to the toxin. Incompatibilities between different genetic codes may have contributed to reproductive isolation among the clades that emerged shortly after point X.

We cannot tell if the hypothesized VLE still exists or was transient. Stochastic losses of infection could explain how ancestral tL CAG genes survive in a few species. If our hypothesis is correct, reorganization of the genetic code can be regarded as a radical mechanism of host defense against an infectious agent 32 , The phylogenetic tree in Fig.

We used RAxML 36 version 8. Branch support for each internode was evaluated with rapid bootstrapping replicates using RAxML Tryptic peptides were resuspended in 0. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. In a first approach to empirical genetic code determination Supplementary Fig.

Settings were Parent Mass Error Tolerance If a genomic site mapped to multiple peptides, all peptides were required to agree. This method deduced the complete genetic code table of each species Supplementary Data 3 , except for ambiguity of Leu and Ile, which cannot be differentiated by mass, and showed that no species had reassigned any codon other than CUG.

In a second approach Supplementary Fig. We then used MaxQuant 43 , 44 version 1. Therefore, we tabulated for each species, for all the CUG sites in its genome that were spanned by a peptide, the proportion of those sites that matched Ser, Ala, Leu, etc.

Genomic DNA of S. RNA was extracted from log-phase cultures by hot acid phenol-chloroform extraction. Primer sequences are listed in Supplementary Table 6. The genome sequences of the other 47 species analyzed are from public sources Supplementary Table 7. Crick, F. The origin of the genetic code. Koonin, E. Origin and evolution of the universal genetic code. Sengupta, S. Pathways of genetic code evolution in ancient and modern organisms.

Keeling, P. Genomics: evolution of the genetic code. Ling, J. Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology. Kollmar, M.

Nuclear codon reassignments in the genomics era and mechanisms behind their evolution. Bioessays 39 , Article Google Scholar. Kawaguchi, Y. The codon CUG is read as serine in an asporogenic yeast Candida cylindracea. Nature , — Ohama, T. Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Res. Butler, G. Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Moura, G. Development of the genetic code: insights from a fungal codon reassignment. FEBS Lett. A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes. Genome Res. Riley, R. Comparative genomics of biotechnologically important yeasts. Natl Acad. USA , — Schultz, D. On malleability in the genetic code. Suzuki, T. EMBO J. Gomes, A. A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans.

Genome Biol. How tRNAs dictate nuclear codon reassignments: only a few can capture non-cognate codons. RNA Biol. Yona, A. Elife 2 , e Rogers, H. RNA 20 , — Sugita, T. Non-universal usage of the leucine CUG codon and the molecular phylogeny of the genus Candida. Microbiol 22 , 79—86 Shen, X. Reconstructing the backbone of the Saccharomycotina yeast phylogeny using genome-scale data. G3 6 , — Yokogawa, T.

USA 89 , — Bezerra, A. Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification. Weil, T. Adaptive mistranslation accelerates the evolution of fluconazole resistance and induces major genomic and gene expression alterations in Candida albicans. Universal rules and idiosyncratic features in tRNA identity. Still other polypeptides must have specific sections removed through a process called proteolysis.

Often, this involves the excision of the first amino acid in the chain usually methionine, as this is the particular amino acid indicated by the start codon. Once a protein is complete, it has a job to perform. Some proteins are enzymes that catalyze biochemical reactions.

Other proteins play roles in DNA replication and transcription. Yet other proteins provide structural support for the cell, create channels through the cell membrane, or carry out one of many other important cellular support functions. This page appears in the following eBook.

Aa Aa Aa. The ribosome assembles the polypeptide chain. What is the genetic code? More on translation. How did scientists discover how ribosomes work?

What are ribosomes made of? Is prokaryotic translation different from eukaryotic translation? Figure 1: In mRNA, three-nucleotide units called codons dictate a particular amino acid.

For example, AUG codes for the amino acid methionine beige. The codon AUG codes for the amino acid methionine beige sphere. The codon GUC codes for the amino acid valine dark blue sphere. The codon AGU codes for the amino acid serine orange sphere. The codon CCA codes for the amino acid proline light blue sphere. The codon UAA is a stop signal that terminates the translation process. The idea of codons was first proposed by Francis Crick and his colleagues in During that same year, Marshall Nirenberg and Heinrich Matthaei began deciphering the genetic code, and they determined that the codon UUU specifically represented the amino acid phenylalanine.

Following this discovery, Nirenberg, Philip Leder, and Har Gobind Khorana eventually identified the rest of the genetic code and fully described which codons corresponded to which amino acids.

Reading the genetic code. Redundancy in the genetic code means that most amino acids are specified by more than one mRNA codon. Methionine is specified by the codon AUG, which is also known as the start codon. Consequently, methionine is the first amino acid to dock in the ribosome during the synthesis of proteins. Tryptophan is unique because it is the only amino acid specified by a single codon. The remaining 19 amino acids are specified by between two and six codons each.

Figure 2 shows the 64 codon combinations and the amino acids or stop signals they specify. Figure 2: The amino acids specified by each mRNA codon. Multiple codons can code for the same amino acid. Figure Detail.

What role do ribosomes play in translation? As previously mentioned, ribosomes are the specialized cellular structures in which translation takes place. This means that ribosomes are the sites at which the genetic code is actually read by a cell.

Figure 3: A tRNA molecule combines an anticodon sequence with an amino acid. These nucleotides represent the anticodon sequence. The nucleotides are composed of a ribose sugar, which is represented by grey cylinders, attached to a nucleotide base, which is represented by a colored, vertical rectangle extending down from the ribose sugar. The color of the rectangle represents the chemical identity of the base: here, the anticodon sequence is composed of a yellow, green, and orange nucleotide.

At the top of the T-shaped molecule, an orange sphere, representing an amino acid, is attached to the amino acid attachment site at one end of the red tube. During translation, ribosomes move along an mRNA strand, and with the help of proteins called initiation factors, elongation factors, and release factors, they assemble the sequence of amino acids indicated by the mRNA, thereby forming a protein.

In order for this assembly to occur, however, the ribosomes must be surrounded by small but critical molecules called transfer RNA tRNA. Each tRNA molecule consists of two distinct ends, one of which binds to a specific amino acid, and the other which binds to a specific codon in the mRNA sequence because it carries a series of nucleotides called an anticodon Figure 3.

In this way, tRNA functions as an adapter between the genetic message and the protein product. The exact role of tRNA is explained in more depth in the following sections. What are the steps in translation? Like transcription, translation can also be broken into three distinct phases: initiation, elongation, and termination.

All three phases of translation involve the ribosome, which directs the translation process. Multiple ribosomes can translate a single mRNA molecule at the same time, but all of these ribosomes must begin at the first codon and move along the mRNA strand one codon at a time until reaching the stop codon. This group of ribosomes, also known as a polysome , allows for the simultaneous production of multiple strings of amino acids, called polypeptides , from one mRNA.

When released, these polypeptides may be complete or, as is often the case, they may require further processing to become mature proteins. Figure 5: To complete the initiation phase, the tRNA molecule that carries methionine recognizes the start codon and binds to it. The bases are represented by blue, orange, yellow, or green vertical rectangles that protrude from the backbone in an upward direction.

Inside the large subunit, the three leftmost terminal nucleotides of the mRNA strand are bound to three anticodon nucleotides in a tRNA molecule. An orange sphere, representing an amino acid, is attached to one tRNA terminus at the top of the molecule. The ribosome is depicted as a translucent complex bound to fifteen nucleotides at the leftmost terminus of the mRNA strand. The tRNA at left has two amino acids attached at its topmost terminus, or amino acid binding site.

The adjacent tRNA at right has a single amino acid attached at its amino acid binding site. A third tRNA molecule is leaving the binding site after having connected its amino acid to the growing peptide chain. There are five additional tRNA molecules with anticodons and amino acids ready to bind to the mRNA sequence to continue to grow the peptide chain. Figure 7: Each successive tRNA leaves behind an amino acid that links in sequence.

The resulting chain of amino acids emerges from the top of the ribosome. The ribosome is depicted as a translucent complex bound to eighteen nucleotides in the middle of the mRNA strand. The tRNA at left has five amino acids attached at its amino acid binding site, forming a chain. Two additional tRNA molecules, each with a single amino acid attached to the amino acid binding site, are approaching the ribosome from the cytoplasm. Figure 8: The polypeptide elongates as the process of tRNA docking and amino acid attachment is repeated.

The ribosome is depicted as a translucent complex bound to many nucleotides at the rightmost terminus of the mRNA strand. A chain of 19 amino acids is attached to the amino acid binding site at the top of the tRNA molecule. The chain is long enough that it extends beyond the upper border of the ribosome and into the cytoplasm.

In the cytoplasm, the peptide chain has folded in on itself several times to form three compact rows of amino acids. Eventually, after elongation has proceeded for some time, the ribosome comes to a stop codon, which signals the end of the genetic message.