Pyl is genetically encoded via an in-frame amber UAG codon, which is normally used as a stop codon to terminate protein synthesis. The PylRS-tRNA Pyl pair has been studied extensively; it is frequently utilized as a tool for genetic code expansion due to its ability to charge a wide variety of non-canonical amino acids ncAAs as well as its orthogonality in both bacterial and eukaryotic hosts Wan et al. The organization of these domains varies between organisms. On the other hand, Pyl-utilizing bacteria such as Desulfitobacterium hafniense encode two individual proteins, PylSc and PylSn, for each domain Nozawa et al.
Finally, seventh-order methanogens such as Methanomethylophilus alvus encode a protein homologous to PylSc, but no homolog of PylSn exists in these archaea Borrel et al. Figure 7. Domain organization and binding mode of PylRS. PylRS is either composed of a fusion of these two domains, two standalone proteins, or as a lone PylSc.
PylSn forms a tight interaction with the variable arm. The distinguishing features of tRNA Pyl are the three-nucleotide variable arm, an elongated anticodon stem from 5 to 6 bp , and a CUA anticodon. More specifically, the universal tRNA Pyl identity elements are the discriminator base G73, and the first bp in the acceptor stem G1:C72 Ambrogelly et al. The M. However, the connecting nucleotide is a U, consistent with the highly conserved U8 in canonical tRNAs.
Furthermore, the D-loop is small, with only five nucleotides, and lacking the widely conserved G18, G19 sequence motif. Details on the identity elements of Mb tRNA Pyl were elucidated by screening its amber suppression efficiency Ambrogelly et al. This study revealed that the nucleotides adjacent to the anticodon U33 and A37, and the T-stem bp GC63 are identity elements.
Figure 8. Identity elements for each tRNA Pyl are highlighted by magenta boxes. The crystal structure of D. The crystal structure of M. Addition of a fourth nucleotide to the variable arm of Mb tRNA Pyl significantly decreases its suppression efficiency, providing further evidence that the interaction between PylRS and the variable arm is critical for aminoacylation Ambrogelly et al.
In canonical tRNAs, this position is widely conserved as U8, which stabilizes tertiary structure through base pairing with A The crystal structure of the D. This also enables G8 to serve as an identity element for the interaction with PylSc, specifically through interaction with residues Arg, Arg, and Glu Herring et al.
Although in vitro aminoacylation assays indicate that the nucleotides flanking the anticodon U33 and A37 are identity elements for Mb PylRS Ambrogelly et al. This desirable trait allowed for general codon reassignment, and thus opened the door for synthetic biologists to incorporate multiple ncAAs into a single protein using different PylRS-tRNA Pyl pairs Wan et al. Other seventh order methanogens such as Methanomassiliicoccus intestinalis and Methanomassiliicoccus lumenyensis tRNA Pyl feature larger breaks that form small loops within the anticodon stem Borrel et al.
An additional difference of M. On the surface, the break in the base pairing of the anticodon stem as well as the lack of a connecting base between the acceptor and D-stem profile as potential identity elements for Ma tRNA Pyl.
Interestingly, deletion of the unpaired nucleotide in the anticodon stem did not significantly alter the translation efficiency of Ma PylRS-tRNA Pyl in a cell-free translation system Yamaguchi et al. Insertion of a C or U between the acceptor and D-stem position 8 moderately decreased translation, but inserting an A or G had no effect Yamaguchi et al. This indicates that the absence of a base in this position may not be an identity element for Ma tRNA Pyl.
Therefore, in this system, the functional role, if any exists, of these unique features of Ma tRNA Pyl is unclear.
Unlike M. Mitochondria are responsible for energy production in eukaryotic cells. As a semi-autonomous organelle descendent from bacteria, mitochondria have their own genome. Mitochondrial genomes not only encode proteins essential for energy production, but also encode parts of the translation machinery, including mitochondrial tRNAs mt-tRNAs Gray et al.
The number of mt-tRNA genes encoded in the mitochondrion varies between organisms. Although canonical tRNAs require conserved structural elements for proper folding, many mt-tRNAs possess highly unusual secondary structures that deviate greatly from canonical tRNAs. Most tRNAs found in organisms are type 0 tRNAs, which have a conserved cloverleaf structure and fold into a tertiary L-shape due to interactions between the D- and T-loops.
On the other hand, mt-tRNAs can be classified into three types based off of their secondary structure Watanabe, ; Suzuki et al. Type I mt-tRNAs have an atypical anticodon stem. Both tRNA structures have only a single nucleotide separating the acceptor and D-stem, have smaller than normal D-loops, elongated anticodon stems, and variable arms consisting of only three nucleotides. Instead, interactions occur between the D-loop and the variable stem to stabilize the mt-tRNA tertiary structure Wakita et al.
Despite lacking a D-stem, this mt-tRNA is functional in vitro and adopts a conformation that is suitable for the ribosome Hanada et al. Figure 9. Mammalian mt-tRNA can be classified into three types.
Instead, the connecting region between the acceptor and anticodon stem interacts with the variable and T-loop to promote folding. Interestingly, mammalian mt-aaRSs appear to have evolved relaxed specificity for their cognate tRNAs.
Specifically, bovine mt-aaRSs have been shown to acylate the corresponding E. Mt-SerRS is even more promiscuous, as it serylates several E. Taken together, these findings indicate that in mammals, mt-aaRSs do not strongly discriminate against non-cognate tRNAs. This apparent lack of specificity may be attributed to the high substrate diversity of mt-tRNAs, or possibly a lack of evolutionary pressure due to the smaller pool of mt-tRNAs present in the cell.
Like their mammalian counterparts, nematode mt-tRNAs have unusual structural features that are distinct from canonical tRNAs. Nematodes encode short mt-tRNAs with diverse cloverleaf structures. Despite greatly deviating from the canonical tRNA cloverleaf structure, evidence suggests that mt-tRNAs lacking one or both sidearms can still interact with tRNA processing enzymes such as CCA-adding enzyme, and are aminoacylated by their cognate synthetases Wolfson et al.
The short T-arm in A. In addition to these observations, recent structural data also indicate that R. Thus, evidence suggests that the D- and T-arms are not required for tRNA to fold into a tertiary conformation suitable for enzymatic activity, and the flexibility of these truncated mt-tRNAs helps to achieve functionality. Figure Nematode mt-tRNAs have diverse and highly unusual secondary structures. Examples of these abnormal mt-tRNA structures are shown here.
In addition to the flexible tertiary structure discussed above, post-transcriptional modifications appear to play an important role in stabilizing mt-tRNAs. Many unmodified mt-tRNAs will not fold properly, but proper modification allows folding and interaction with tRNA processing enzymes to occur Lorenz et al. For instance, 1-methyl adenosine at position 9 m 1 A9 is found in many mt-tRNA species, including those lacking one or both sidearms, and this modification is important for proper cloverleaf folding to occur Helm et al.
Nematode mt-tRNA lacking the D-arm possess m 1 A9 as well as several pseudouridine in the acceptor and anticodon stem Sakurai et al. Ultimately, post-translational modification appears to play an important role in facilitating mt-tRNA activity and stability, including the truncated mt-tRNAs that lack a canonical cloverleaf structure. Deviations from the standard genetic code have been reported in the mitochondria of green plant algae from the phylum Chlorophyta Noutahi et al.
Similar to the pyrrolysine incorporation system in archaea and bacteria, the stop codons UAG and UGA are reassigned to sense codons in some Chlorophyta.
Recent evidence suggests that in addition to stop codon reassignment, sense codons may also be reassigned in green algae. Sense codon reassignment has also been observed in S. The observation that codon reassignment occurs in mitochondria across kingdoms underscores the dynamic nature of the mitochondrial genome.
The unique structures found in these non-canonical tRNAs appear to be a result of their necessary function and the enzymes that they interact with. Despite deviating from the canonical structure, majority of the tRNAs presented in this review have been found to be functional in translation.
In mitochondria, highly unusual mt-tRNAs with diverse structures are used along with specialized mitochondrial translation machinery to translate proteins encoded by the mitochondrial genome Gray et al. The translational machinery has evolved to accept a wide variety of tRNA structures for efficient translation of proteins in the desired host. A significant amount of effort has been put forth to expand the genetic code, pushing the boundaries of what functionality can be incorporated into proteins.
To that end, engineered aaRS-tRNA pairs have been utilized to incorporate numerous diverse ncAAs into proteins both in vitro and in vivo. This plasticity may indicate a lack of evolutionary pressure to discriminate against unknown or unusual tRNAs that are rarely, if ever, encountered by the host cell.
Thus, it is plausible that the unusual structures of specialized or non-canonical tRNAs such as the ones described in this review are made possible by a lack of evolutionary pressure to maintain the canonical structure. An alternative possibility is that many of these non-canonical tRNAs originated from an ancient, more diverse genetic code, and because of their specialized and infrequent usage, they were never pressured to evolve into a canonical tRNA structure.
In either case, if deviations from the canonical tRNA structure are well-tolerated by the aminoacyl-synthetase and translation machinery, mutations or structural changes to the tRNA can potentially occur without consequence and lead to polymorphisms over time. This can be seen in mitochondria. Highly variable mt-tRNAs are well-known to be susceptible to mutations, and while mt-tRNA mutations to critical nucleotides can cause diseases, neutral or slightly deleterious polymorphisms frequently occur and are inconsequential Lynch, ; Wittenhagen and Kelley, ; Yarham et al.
Ultimately, despite their many differences from the canonical tRNA structure, non-canonical tRNAs are readily utilized in translation and enable the cell to produce proteins that are, in many cases, essential for survival Longstaff et al. NK and JF wrote the manuscript. DS edited the manuscript.
All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Alfonzo, J. Mitochondrial tRNA import—the challenge to understand has just begun.
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Essays Biochem. Dudek, J. The loop closest to the 3' end is called the T arm, after its sequence of thymine-pseudouridine-cytosine pseudouridine is also an unusual base. The loop on the bottom of the cloverleaf contains the anticodon, which binds complementarily to the mRNA codon. Because anticodons bind with codons in antiparallel fashion, they are written from the 5' end to 3' end, the inverse of codons.
For example, the anticodon in the figure above should be written 3'-CGU-5'. At the 3' end of the tRNA molecule, opposite the anticodon, extends a three nucleotide acceptor site that includes a free -OH group. A specific tRNA binds to a specific amino acid through its acceptor stem.
The cloverleaf structure shown above is actually a two dimensional simplification of the actual tRNA structure. The cloverleaf is therefore called a secondary structure. In reality, the cloverleaf folds further into a tertiary structure, a sort of vague L-shape.
Each tRNA can be linked to an amino acid. The tRNA has an anticodon sequence that complementary to a triplet codon representing the amino acid. The anticodon is a trinucleotide sequence in tRNA which is complementary to the codon in mRNA and enables the tRNA to place the appropriate amino acid in response to the codon. The cloverleaf describes the structure of tRNA drawn in two dimensions, forming four distinct arm-loops.
A stem is the base-paired segment of a hairpin structure in RNA. An arm of tRNA is one of the four or in some cases five stem-loop structures that make up the secondary structure. The anticodon arm of tRNA is a stem loop structure that exposes the anticodon triplet at one end. The D arm of tRNA has a high content of the base dihydrouridine. It is the most variable in length in tRNA, from bases. Conserved positions are defined when many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations.
A semiconserved Semiinvariant position is one where comparison of many individual sequences finds the same type of base pyrimidine or purine always present. A tRNA has a sequence of bases that folds into a clover-leaf secondary structure with four constant arms and an additional arm in the longer tRNAs. The sequence of the anticodon is solely responsible for the specificity of the aminoacyl-tRNA. Messenger RNA can be distinguished from the apparatus responsible for its translation by the use of in vitro cell-free systems to synthesize proteins.
A protein-synthesizing system from one cell type can translate the mRNA from another, demonstrating that both the genetic code and the translation apparatus are universal. Each nucleotide triplet in the mRNA represents an amino acid.
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