Codon-anticodon Pairing Research Articles

Maximal Genetic Code Symmetry Is a Physicochemical Purine-Pyrimidine Symmetry Language for Transcription and Translation in the Flow of Genetic Information from DNA to Proteins.

Until now, research has not taken into consideration the physicochemical purine-pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine-pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson-Crick and codon-anticodon pairing. The maximal purine-pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson-Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine-pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis.

A tRNA modification pattern that facilitates interpretation of the genetic code.

Interpretation of the genetic code from triplets of nucleotides to amino acids is fundamental to life. This interpretation is achieved by cellular tRNAs, each reading a triplet codon through its complementary anticodon (positions 34-36) while delivering the amino acid charged to its 3'-end. This amino acid is then incorporated into the growing polypeptide chain during protein synthesis on the ribosome. The quality and versatility of the interpretation is ensured not only by the codon-anticodon pairing, but also by the post-transcriptional modifications at positions 34 and 37 of each tRNA, corresponding to the wobble nucleotide at the first position of the anticodon and the nucleotide on the 3'-side of the anticodon, respectively. How each codon is read by the matching anticodon, and which modifications are required, cannot be readily predicted from the codon-anticodon pairing alone. Here we provide an easily accessible modification pattern that is integrated into the genetic code table. We focus on the Gram-negative bacterium Escherichia coli as a model, which is one of the few organisms whose entire set of tRNA modifications and modification genes is identified and mapped. This work provides an important reference tool that will facilitate research in protein synthesis, which is at the core of the cellular life.

Critical cis-parameters influence STructure assisted RNA translation (START) initiation on non-AUG codons in eukaryotes.

In eukaryotes, translation initiation is a highly regulated process, which combines cis-regulatory sequences located on the messenger RNA along with trans-acting factors like eukaryotic initiation factors (eIF). One critical step of translation initiation is the start codon recognition by the scanning 43S particle, which leads to ribosome assembly and protein synthesis. In this study, we investigated the involvement of secondary structures downstream the initiation codon in the so-called START (STructure-Assisted RNA translation) mechanism on AUG and non-AUG translation initiation. The results demonstrate that downstream secondary structures can efficiently promote non-AUG translation initiation if they are sufficiently stable to stall a scanning 43S particle and if they are located at an optimal distance from non-AUG codons to stabilize the codon-anticodon base pairing in the P site. The required stability of the downstream structure for efficient translation initiation varies in distinct cell types. We extended this study to genome-wide analysis of functionally characterized alternative translation initiation sites in Homo sapiens. This analysis revealed that about 25% of these sites have an optimally located downstream secondary structure of adequate stability which could elicit START, regardless of the start codon. We validated the impact of these structures on translation initiation for several selected uORFs.

Posttranscriptional modification to the core of tRNAs modulates translational misreading errors.

Protein synthesis on the ribosome involves successive rapid recruitment of cognate aminoacyl-tRNAs and rejection of the much more numerous incorrect near- or non-cognates. The principal feature of translation elongation is that at every step, many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translational accuracy require that cognate tRNAs have relatively similar acceptance rates by the ribosome. To achieve that, tRNAs evolved to compensate for differences in amino acid properties and codon-anticodon strength that affect acceptance. Part of that response involved tRNA posttranscriptional modifications, which can affect tRNA decoding efficiency, accuracy, and structural stability. The most intensively modified regions of the tRNA are the anticodon loop and structural core of the tRNA. Anticodon loop modifications directly affect codon-anticodon pairing and therefore modulate accuracy. Core modifications have been thought to ensure consistent decoding rates principally by stabilizing tRNA structure to avoid degradation; however, degradation due to instability appears to only be a significant issue above normal growth temperatures. We suspected that the greater role of modification at normal temperatures might be to tune tRNAs to maintain consistent intrinsic rates of acceptance and peptide transfer and that hypomodification by altering these rates might degrade the process of discrimination, leading to increased translational errors. Here, we present evidence that most tRNA core modifications do modulate the frequency of misreading errors, suggesting that the need to maintain accuracy explains their deep evolutionary conservation.

Modulation of translational decoding by m6A modification of mRNA

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life.

The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon-codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3-/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD-TsaB -TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway-Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure-function relationship from perspectives of conservation and diversity.

Multiplex suppression of four quadruplet codons via tRNA directed evolution

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing.

Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguanosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop andimmediately adjacent to the anticodon nucleotides 34, 35, 36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and the lack of m1G37 destabilize interactions of tRNAPro with the P site of the ribosome, causing large conformational changes typically only seen during EF-G-mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome in the context of a slippery mRNA codon.

Mutations in domain IV of elongation factor EF-G confer -1 frameshifting.

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon–anticodon pairing and slippage of the reading frame by −1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon–anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased −1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.

Aspects of Nucleic Acid Structure and Function

Despite decades of intense study, certain physico-chemical aspects of the nucleic acids–the genetic repository of life–remain enigmatic. Thus, solutions of DNA are apparently constituted of varying amounts of double and single-stranded forms, with melting studies being inconclusive about the effect of temperature on the composition. Consequently, this casts doubt on current estimates of the thermodynamic stability of the base pairs. However, the overwhelming stability of the Watson-Crick model is adumbrated by a kinetic analysis of the action of DNA polymerase, microscopic reversibility indicating that polymerase action is kinetically controlled, whereas proof reading-excision is thermodynamically controlled.
 The structural basis for the differing roles played by DNA and RNA in the sustenance and propagation of life also remains to be clarified. It appears that formation of the RNA double helix is sterically inhibited by the 2’ ribose hydroxyl group, which is also relatively inaccessible to external base, thus enhancing hydrolytic stability. The in vivo conformation of RNA is likely determined by the requirements of its appointed biological role, the large size of tRNAs being particularly significant in possibly leading to enhanced specificity in its interaction with the corresponding tRNA synthetase. The proclivity of RNA generally to remain single stranded may indeed be the reason for the existence of viruses as stable RNA-protein complexes.
 The preeminent role of nucleic acids as “genetic guardians” is, however, blurred by the fact that the translation of the genetic code is contingent on the action of the various tRNA synthetases. This implies that the genetic code is manifested via codon-anticodon specificity only by the involvement of protein molecules that are unique to each codon-anticodon pair. This has intriguing implications for the origin and evolution of the genetic code, possibly indicating that the tRNA synthetases are a relic of prebiotic protein-nucleic acid hybrids (thus also raising doubts about the RNA world hypothesis).

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons.

We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid synthesizes proteins containing the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon-anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and, in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.

Codon-Specific Translation by m1G37 Methylation of tRNA.

Although the genetic code is degenerate, synonymous codons for the same amino acid are not translated equally. Codon-specific translation is important for controlling gene expression and determining the proteome of a cell. At the molecular level, codon-specific translation is regulated by post-transcriptional epigenetic modifications of tRNA primarily at the wobble position 34 and at position 37 on the 3′-side of the anticodon. Modifications at these positions determine the quality of codon-anticodon pairing and the speed of translation on the ribosome. Different modifications operate in distinct mechanisms of codon-specific translation, generating a diversity of regulation that is previously unanticipated. Here we summarize recent work that demonstrates codon-specific translation mediated by the m1G37 methylation of tRNA at CCC and CCU codons for proline, an amino acid that has unique features in translation.

Ribosomal ambiguity(ram) mutations promote the open (off) to closed (on) transition and thereby increase miscoding.

Decoding is thought to be governed by a conformational transition in the ribosome—open (off) to closed (on)—that occurs upon codon–anticodon pairing in the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to disparate regions, consistent with a role for ribosome dynamics in decoding, yet precisely how these mutations act has been unclear. Here, we solved crystal structures of 70S ribosomes harboring 16S ram mutations G299A and G347U in the absence A-site tRNA (A-tRNA) and in the presence of a near-cognate anticodon stem-loop (ASL). In the absence of an A-tRNA, each of the mutant ribosomes exhibits a partially closed (on) state. In the 70S-G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S-G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed (on) conformation in the presence of near-cognate A-tRNA, just as they do with cognate A-tRNA. Thus, these ram mutations act by promoting the open (off) to closed (on) transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder rotation for productive aminoacylated-tRNA incorporation.

Interaction of rRNA with mRNA and tRNA in Translating Mammalian Ribosome: Functional Implications in Health and Disease

RNA-RNA interaction slowly emerges as a critical component for the smooth functioning of gene expression processes, in particular in translation where the central actor is an RNA powered molecular machine. Overall, ribosome dynamic results from sequential interactions between three main RNA species: ribosomal, transfer and messenger RNA (rRNA, tRNA and mRNA). In recent decades, special attention has been paid to the physical principles governing codon-anticodon pairing, whereas individual RNA positioning mostly relies on ribosomal RNA framework. Here, we provide a brief overview on the actual knowledge of RNA infrastructure throughout the process of translation in mammalian cells: where and how do these physical contacts occur? What are their potential roles and functions? Are they involved in disease development? What will be the main challenges ahead?

Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases

Class I and II aaRS recognition of opposite grooves was likely among the earliest determinants fixed in the tRNA acceptor stem bases. A new regression model identifies those determinants in bacterial tRNAs. Integral coefficients relate digital dependent to independent variables with perfect agreement between observed and calculated grooves for all twenty isoaccepting tRNAs. Recognition is mediated by the Discriminator base 73, the first base pair, and base 2 of the acceptor stem. Subsets of these coefficients also identically compute grooves recognized by smaller numbers of aaRS. Thus, the model is hierarchical, suggesting that new rules were added to pre-existing ones as new amino acids joined the coding alphabet. A thermodynamic rationale for the simplest model implies that Class-dependent aaRS secondary structures exploited differential tendencies of the acceptor stem to form the hairpin observed in Class I aaRS•tRNA complexes, enabling the earliest groove discrimination. Curiously, groove recognition also depends explicitly on the identity of base 2 in a manner consistent with the middle bases of the codon table, confirming a hidden ancestry of codon-anticodon pairing in the acceptor stem. That, and the lack of correlation with anticodon bases support prior productive coding interaction of tRNA minihelices with proto-mRNA.

How 2'-O-Methylation in mRNA Disrupts tRNA Decoding during Translation Elongation

Chemical modifications of messenger RNA (mRNA) may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation (Nm) of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in certain amino acid codons. Here, using single-molecule, bulk kinetics and structural methods, we show that Nm within coding regions of mRNA disrupts key steps in codon reading during cognate transfer RNA (tRNA) selection. Our results suggest that Nm sterically perturbs interactions of ribosomal monitoring bases (G530, A1492 and A1493 of 16S ribosomal RNA) with cognate codon-anticodon helices, thereby inhibiting downstream GTP-hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated-tRNAs in initial selection and proofreading. The observed effect of monitoring bases in decoding accuracy mirrors the effect of disturbed codon-anticodon pairing, previously studied using N6-methyladenoside (m6A) modified mRNA. Combined together, our findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.