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Codon-reassignment in the Escherichia coli genetic code. RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites. 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. As a result, the ribosome detaches from the mRNA and releases the amino acid chain.
This marks the final phase of translation, which is called termination Figure 9. Figure 9: The translation process terminates after a stop codon signals the ribosome to fall off the RNA. Each subunit exists separately in the cytoplasm, but the two join together on the mRNA molecule.
The tRNA molecules are adaptor molecules—they have one end that can read the triplet code in the mRNA through complementary base-pairing, and another end that attaches to a specific amino acid Chapeville et al.
The idea that tRNA was an adaptor molecule was first proposed by Francis Crick, co-discoverer of DNA structure, who did much of the key work in deciphering the genetic code Crick, The rRNA catalyzes the attachment of each new amino acid to the growing chain. Interestingly, not all regions of an mRNA molecule correspond to particular amino acids. In particular, there is an area near the 5' end of the molecule that is known as the untranslated region UTR or leader sequence. This portion of mRNA is located between the first nucleotide that is transcribed and the start codon AUG of the coding region, and it does not affect the sequence of amino acids in a protein Figure 3.
So, what is the purpose of the UTR? It turns out that the leader sequence is important because it contains a ribosome-binding site. A similar site in vertebrates was characterized by Marilyn Kozak and is thus known as the Kozak box. If the leader is long, it may contain regulatory sequences, including binding sites for proteins, that can affect the stability of the mRNA or the efficiency of its translation.
Figure 4: The translation initiation complex. When translation begins, the small subunit of the ribosome and an initiator tRNA molecule assemble on the mRNA transcript. The small subunit of the ribosome has three binding sites: an amino acid site A , a polypeptide site P , and an exit site E.
Here, the initiator tRNA molecule is shown binding after the small ribosomal subunit has assembled on the mRNA; the order in which this occurs is unique to prokaryotic cells.
In eukaryotes, the free initiator tRNA first binds the small ribosomal subunit to form a complex. Figure Detail Although methionine Met is the first amino acid incorporated into any new protein, it is not always the first amino acid in mature proteins—in many proteins, methionine is removed after translation.
In fact, if a large number of proteins are sequenced and compared with their known gene sequences, methionine or formylmethionine occurs at the N-terminus of all of them. However, not all amino acids are equally likely to occur second in the chain, and the second amino acid influences whether the initial methionine is enzymatically removed. For example, many proteins begin with methionine followed by alanine. In both prokaryotes and eukaryotes, these proteins have the methionine removed, so that alanine becomes the N-terminal amino acid Table 1.
However, if the second amino acid is lysine, which is also frequently the case, methionine is not removed at least in the sample proteins that have been studied thus far. These proteins therefore begin with methionine followed by lysine Flinta et al.
Table 1 shows the N-terminal sequences of proteins in prokaryotes and eukaryotes, based on a sample of prokaryotic and eukaryotic proteins Flinta et al. In the table, M represents methionine, A represents alanine, K represents lysine, S represents serine, and T represents threonine.
Once the initiation complex is formed on the mRNA, the large ribosomal subunit binds to this complex, which causes the release of IFs initiation factors.
The large subunit of the ribosome has three sites at which tRNA molecules can bind. The A amino acid site is the location at which the aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that correct amino acid is added to the growing polypeptide chain. The P polypeptide site is the location at which the amino acid is transferred from its tRNA to the growing polypeptide chain.
Finally, the E exit site is the location at which the "empty" tRNA sits before being released back into the cytoplasm to bind another amino acid and repeat the process.
The ribosome is thus ready to bind the second aminoacyl-tRNA at the A site, which will be joined to the initiator methionine by the first peptide bond Figure 5. Figure 5: The large ribosomal subunit binds to the small ribosomal subunit to complete the initiation complex.
The initiator tRNA molecule, carrying the methionine amino acid that will serve as the first amino acid of the polypeptide chain, is bound to the P site on the ribosome. The A site is aligned with the next codon, which will be bound by the anticodon of the next incoming tRNA.
Next, peptide bonds between the now-adjacent first and second amino acids are formed through a peptidyl transferase activity. For many years, it was thought that an enzyme catalyzed this step, but recent evidence indicates that the transferase activity is a catalytic function of rRNA Pierce, After the peptide bond is formed, the ribosome shifts, or translocates, again, thus causing the tRNA to occupy the E site.
The tRNA is then released to the cytoplasm to pick up another amino acid. In addition, the A site is now empty and ready to receive the tRNA for the next codon. This process is repeated until all the codons in the mRNA have been read by tRNA molecules, and the amino acids attached to the tRNAs have been linked together in the growing polypeptide chain in the appropriate order. At this point, translation must be terminated, and the nascent protein must be released from the mRNA and ribosome.
To ensure that the presence of the in-frame UAG codons at these hyperconserved sites was supported by original sequencing data, we inspected raw RNA-seq reads mapped onto the respective contigs. No conflicting signal concerning the identity of the nucleotides corresponding to any of these UAG codons was apparent each in-frame UAG was supported by more than one read, with the read variability lower than 4.
As an alternative to the hyperconserved position-based inference of the UAG codon meaning, we devised a phylogeny-informed ML-based method that unselectively considers all UAG positions see Methods for details.
Briefly, we first inferred an organismal phylogeny using a smaller dataset of eight conserved proteins to save computation time , with the respective genes from the rhizarian exLh containing 71 in-frame UAG codons, represented as an undetermined amino acid X in the alignment. We then prepared 20 modifications of the dataset, each with a different amino acid considered at positions corresponding to the in-frame UAG codons in the genes from the rhizarian exLh.
Then, we calculated the best ML tree for each of the 20 datasets, using the same substitution model and the tree from the initial dataset as a constraint. The dataset where UAG was translated as leucine showed the highest likelihood score, and the conditional probability that UAG encodes leucine in the rhizarian exLh calculated conditional upon UAG encoding one amino acid at all positions is virtually 1.
Based on these results, we conclude that UAG is the seventh codon for leucine in the rhizarian exLh and does not serve as a stop codon in this organism. This is not without precedent, as the stop-to-leucine UAG reassignment was previously reported from mitochondrial genomes of several green algae of the order Sphaeropleales [ 22 , 28 ] and of the chytrid fungus Spizellomyces punctatus and its relatives [ 21 ].
Interestingly, we noticed striking differences in the UAG codon abundance between certain groups of genes from the rhizarian exLh. In-frame UAG codons were overrepresented in genes encoding components of the 26S proteasome, where the UAG codon was the most abundant codon for leucine Fig.
In contrast, UAG was the rarest leucine codon in genes for ribosomal proteins. Genes for ribosomal proteins are highly expressed and typically exhibit a strong codon usage bias facilitating efficient synthesis of ribosomal proteins [ 29 ]. The low abundance of the UAG codon in ribosomal protein genes in the rhizarian exLh thus suggests that this codon is not as efficiently translated as the six standard codons for leucine. Relative codon frequencies in the rhizarian exLh and I.
The relative codon frequencies are calculated as the percentage of the codon among all occurrences of codons with the same meaning i. The second case of a novel non-canonical genetic code was unexpectedly encountered when we sequenced the transcriptome of I. It is a recently described anaerobic flagellate isolated from fresh feces of a gecko Phelsuma grandis and is considered an intestinal endobiont [ 30 ].
To corroborate this initial insight, we used the data from the newly sequenced transcriptome of I. A protein phylogenomic analysis the same as used above for establishing the phylogenetic position of the rhizarian exLh with the dataset including 60 orthologs from I.
This is consistent with the previous result based on the partial 18S rRNA gene sequence and with the fact that no other non-diplomonad fornicate could be included in the analysis due to lack of genome-scale sequence data.
The second analysis was based on a dataset comprising a complete 18S rRNA sequence, which we identified as one of the assembled transcript contigs, and sequences of four conserved proteins used in a previous detailed study of the phylogeny of the Fornicata [ 32 ]. This analysis placed I. Thus, it is now robustly established that I. In addition, it is a lineage well separated from Hexamitinae, a subgroup of diplomonads, which is a conclusion important for the interpretation of the evolution of the genetic code in fornicates see below.
While analyzing the assembled transcript sequences from I. We used similar approaches as employed for analyzing the genetic code of the rhizarian exLh to determine the identity of this UAG-encoded amino acid see Methods for details.
First, using a smaller subset of genes sampled broadly to include representatives from most major eukaryotic lineages we identified 28 hyperconserved positions with an in-frame UAG in I.
In the second analysis, a concatenated protein sequence alignment considering glutamine in place of in-frame UAG codons in I. To further test that UAG is the only termination codon reassigned in I. In total, we analyzed contigs assigned to I. Although this value may seem low and ambiguous, a similar proportion of conserved alignment positions dominated by glutamine For canonical glutamine codons in I.
In addition, neither of the examined transcripts included the UAG codon as an obvious termination codon marking the end of the coding sequence. All these results indicate that UAG in I. In contrast, our procedure identified only two contigs ac02 and ac03 with candidate in-frame UAA or UGA codons, but manual scrutiny revealed that these codons are located in regions representing obvious retained introns. However, this was an apparent error, as it is beyond any doubt that Blepharisma spp.
Indeed, the most recent list of genetic code tables provided on another NCBI page [ 35 ] omits the code Thus, the code we here document for I. Rhizaria is an extremely diverse eukaryotic grouping, but our knowledge of even the general biology, let alone molecular details, of most of rhizarian groups is lamentable. The discovery of a new rhizarian lineage exhibiting a peculiar feature of their gene expression machinery is thus not so unexpected. Our phylogenetic analyses place the new rhizarian with the stop-to-leucine reassignment of the UAG codon into the group Sainouroidea see above.
We should, therefore, ask how widespread this feature is in Sainouroidea or possibly a broader rhizarian clade. The closest relative of the rhizarian exLh, for which a substantial amount of sequences of protein coding genes transcripts are available, is G.
Brown et al. Therefore, we analyzed the transcript sequences from G. Brown, personal communication. Interestingly, all but one available G. Thus, G. Little data is available on nuclear protein-coding genes of other sainouroids, specifically a single sequence for each of Rosculus sp.
Neither of these sequences exhibits in-frame termination codons, and the gene from Rosculus sp. These sequences are, therefore, consistent with the notion that the genetic code has changed specifically in the rhizarian exLh lineage, but a systematic exploration of sainouroid transcriptomes or genomes is needed to pinpoint this evolutionary event with a higher confidence. While our study uncovers the first case of a non-canonical nuclear code for the whole Rhizaria, the departure from the standard code reported here from I.
Specifically, hexamitin diplomonads Hexamitinae for example, members of the genera Spironucleus , Trimitus or Trepomonas , also encode glutamine by non-standard codons [ 37 , 38 ]. However, in contrast to I. Hexamitins and the I. The closest I. All four these sequences GenBank accession numbers AB It will be interesting not only to obtain more complete data for investigating the genetic code of H.
Let us now touch briefly upon the actual molecular underpinnings of the changed specificity of the UAG codon in the rhizarian exLh and I. Therefore, we predict that sequencing the genomes of the rhizarian exLh and I. Notably, anticodons of these tRNAs differ in only one nucleotide position from anticodons of standard tRNAs carrying the respective amino acids, i.
In addition, leucinyl-tRNA synthetases generally do not recognize the anticodon as a tRNA identity element [ 39 — 41 ], suggesting that efficient charging by leucine of the newly emerged tRNA Leu CUA does not necessarily require changes in the enzyme.
Nevertheless, the multiple independent cases of the stop-to-glutamine UAG reassignment in various eukaryotes Fig. Phylogenetic distribution of known non-canonical genetic codes in nuclear genes of eukaryotes. The schematic phylogenetic tree was drawn on the basis of phylogenetic and phylogenomic analyses for eukaryotes as a whole [ 60 , 71 , 72 ] our own Fig. Multifurcations indicate uncertain or controversial branching order, dashed branches indicate different positions of Metamonada within eukaryotes suggested by different studies, branches drawn as double lines indicate paraphyletic groupings.
The types and occurrences of the different non-canonical codes are based on this study the rhizarian exLh and Iotanema and the following previous reports: fungi [ 14 , 15 ]; Amoeboaphelidium [ 13 ]; oxymonads [ 11 ]; Blastocrithidia [ 18 ]; ulvophytes [ 12 ]; ciliates [ 7 , 9 , 16 , 17 ].
We also omitted some ciliate species with their putative non-canonical codes supported by little data that are specifically related to and possibly sharing the same code with better studied species. Changes in the genetic code are mapped onto the tree primarily black circles using Dollo parsimony no reversions are allowed. An alternative maximum parsimony scenario with reversions weighted the same as other changes is indicated by the respective code numbers in white circles.
An alternative branching order to the one indicated in the figure was supported by some studies for some of the ciliate lineages, but the alternative topology does not decrease the minimal number of codon reassignments required to explain the distribution of non-standard genetic codes. This will require identification and culturing the rhizarian exLh; work towards this goal is underway in our laboratory.
Sequencing the genome of I. Moreover, it should be noted that identification of tRNAs responsible for reading reassigned termination codons may not be straightforward even when the genome sequence is available.
For example, Swart et al. The specificity of tRNAs is not necessarily obvious from the gene sequence itself, as post-transcriptional editing or base modifications may be involved, too.
As a result, the actual tRNAs responsible for termination codon reassignments remain unknown for most of the previously described non-canonical codes in eukaryotic nuclear genomes.
In eukaryotes, translation termination is mediated by the interaction of all three termination codons with the same protein, eRF1 eukaryotic release factor 1 , specifically with its N-terminal domain [ 43 , 44 ].
Indeed, eRF1 sequences in eukaryotes that have altered the meaning of UAG, UAA, or UGA codons proved to typically exhibit various alterations in these motifs when compared to eRF1 sequences from organisms with the canonical code [ 9 , 47 , 48 ], and some of these changes have been demonstrated as causally linked to an altered specificity of the eRF1 protein towards the termination codons [ 45 , 49 ].
We identified transcripts encoding eRF1 in both the rhizarian exLh and I. The eRF1 sequence from the rhizarian exLh does not display any obvious deviation in the conserved elements noticed above, but it notably exhibits an alanine residue at the Leu69 position of the human eRF1 protein.
Although this position is not particularly conserved among eRF1 proteins, the substitution to alanine is unique for the rhizarian exLh Additional file 6 : Figure S2 and a corresponding L69A mutation was shown to increase readthrough of all three stop codons, particularly of the UAG codon, suggesting that this position is specifically important for the recognition of guanine of the third stop codon position by eRF1 [ 45 ].
It is, therefore, possible that this substitution is partly responsible for efficient usage of UAG as a sense codon in the rhizarian exLh. The most conspicuous feature of the eRF1 protein from I. This seems to be significant. With adenosine in the second position of the termination codon, Thr32 faces the base at the third position, making a hydrogen bond with the N2 atom of guanosine in UAG [ 46 ]. Hence, the T32G substitution presumably disrupts this interaction and weakens the affinity of the I.
The analysis of the eRF1 sequences from the rhizarian exLh and I. Since the discovery of the first non-standard genetic code in ciliates more than 30 years ago until our study, all known nuclear genetic code alternations have followed a regular pattern that evolutionary changes in the meaning of the UAG and UAA codons are coupled. We mapped the distribution of documented non-standard genetic codes in eukaryotic nuclear genomes onto the species phylogeny and deduced the most parsimonious scenario explaining the origin of these codes Fig.
The analysis predicts at least 13 independent evolutionary changes in the meaning of coordinately in both UAG and UAA including possible reversions to the standard code or a putative change of an encoded amino acid from glutamine to glutamate in a particular ciliate lineage; Fig.
This is in stark contrast to the situation in mitochondria, which exhibit a plethora of different non-canonical codes, including those with stop-to-sense reassignments of UAG encoding leucine independently in some chytrids and green algae; see above or UAA encoding tyrosine in some flatworms [ 50 ] , but no known case of a simultaneous reassignment of both UAG and UAA.
There might be some inherent molecular predisposition for different evolutionary trajectories of the UAG and UAA codons in nuclear and mitochondrial codes for example, related to the differences in the mechanism of translation termination , but the discovery of the two new code variants in the rhizarian exLh and I. Our study thus provides an important new perspective on the evolution of the genetic code in eukaryotes.
In addition, we developed a new generally applicable phylogeny-informed method for inferring the meaning of reassigned codons that will facilitate characterization of non-standard genetic codes to be discovered in the future. Our research has also contributed to improving our knowledge of the phylogenetic diversity of eukaryotes. The discovery of a new insect-associated lineage of Rhizaria, interesting in itself, may be of a special significance, because the host species, the heteropteran L.
The second subject of our study, I. No genome-level sequence data has been reported so far from non-diplomonad fornicates, so our sequencing of the I. Searching the TSA from L. Three of them GBHO The remaining two contigs, GBHO We hypothesized that it is derived from the same organism as the Rhizaria-related component detected among protein-coding transcripts.
Careful examination of these two contigs by blast searches against the NCBI sequence databases and against the raw Illumina reads from L. The contig GBHO The latter was thus interpreted by us as the actual rRNA locus of L.
Examination of Illumina reads mapped onto this contig and detailed sequence comparisons revealed that the artificial fusion of the two segments was due to the fact that the 18S rRNA from L.
This newly assembled part is for unknown reasons completely missing from the L. Nevertheless, we did not notice any ambiguities in the alignment of RNA-seq reads along the sequence and a large part of the reassembled sequence was confirmed by PCR and sequencing see the next section , corroborating our interpretation of the RNA-seq data.
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