Sense codon reassignment enables viral resistance and encoded polymer synthesis.
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ABSTRACT:
SUBMITTER: Robertson WE
PROVIDER: S-EPMC7611380 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2'-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylalanine. The strategy developed here is generic, as demonstrated by sequestration of tRNAArgCCU isoacceptor in E. coli translation system. Furthermore, this method is likely to be species-independent and was successfully applied to the eukaryotic Leishmania tarentolae in vitro translation system. This approach represents a new direction in genetic code reassignment with numerous practical applications.
| S-EPMC6158751 | biostudies-literature
Project description:BackgroundEfficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature.ResultsIn the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences.ConclusionsWe suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants.
| S-EPMC3161013 | biostudies-literature
Project description:Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNAOpt We suspected a modification of the tRNAOptAUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUG is converted to inosine. We identified tRNAOptAUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.
| S-EPMC5137457 | biostudies-literature
Project description:The extent to which alteration of endogenous tRNA modifications may be exploited to improve genetic code expansion efforts has not been broadly investigated. Modifications of tRNAs are strongly conserved evolutionarily, but the vast majority of E. coli tRNA modifications are not essential. We identified queuosine (Q), a non-essential, hypermodified guanosine nucleoside found in position 34 of the anticodons of four E. coli tRNAs as a modification that could potentially be utilized to improve sense codon reassignment. One suggested purpose of queuosine modification is to reduce the preference of tRNAs with guanosine (G) at position 34 of the anticodon for decoding cytosine (C) ending codons over uridine (U) ending codons. We hypothesized that introduced orthogonal translation machinery with adenine (A) at position 34 would reassign U-ending codons more effectively in queuosine-deficient E. coli. We evaluated the ability of introduced orthogonal tRNAs with AUN anticodons to reassign three of the four U-ending codons normally decoded by Q34 endogenous tRNAs: histidine CAU, asparagine AAU, and aspartic acid GAU in the presence and absence of queuosine modification. We found that sense codon reassignment efficiencies in queuosine-deficient strains are slightly improved at Asn AAU, equivalent at His CAU, and less efficient at Asp GAU codons. Utilization of orthogonal pair-directed sense codon reassignment to evaluate competition events that do not occur in the standard genetic code suggests that tRNAs with inosine (I, 6-deaminated A) at position 34 compete much more favorably against G34 tRNAs than Q34 tRNAs. Continued evaluation of sense codon reassignment following targeted alterations to endogenous tRNA modifications has the potential to shed new light on the web of interactions that combine to preserve the fidelity of the genetic code as well as identify opportunities for exploitation in systems with expanded genetic codes.
| S-EPMC9471426 | biostudies-literature
Project description:The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.
| S-EPMC4652775 | biostudies-literature
Project description:Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code.
| S-EPMC7830368 | biostudies-literature
Project description:The majority of bacteriophage diversity remains uncharacterised, and new intriguing mechanisms of their biology are being continually described. Members of some phage lineages, such as the Crassvirales, repurpose stop codons to encode an amino acid by using alternate genetic codes. Here, we investigated the prevalence of stop codon reassignment in phage genomes and subsequent impacts on functional annotation. We predicted 76 genomes within INPHARED and 712 vOTUs from the Unified Human Gut Virome catalogue (UHGV) that repurpose a stop codon to encode an amino acid. We re-annotated these sequences with modified versions of Pharokka and Prokka, called Pharokka-gv and Prokka-gv, to automatically predict stop codon reassignment prior to annotation. Both tools significantly improved the quality of annotations, with Pharokka-gv performing best. For sequences predicted to repurpose TAG to glutamine (translation table 15), Pharokka-gv increased the median gene length (median of per genome medians) from 287 to 481 bp for UHGV sequences (67.8% increase) and from 318 to 550 bp for INPHARED sequences (72.9% increase). The re-annotation increased mean coding density from 66.8% to 90.0%, and from 69.0% to 89.8% for UHGV and INPHARED sequences. Furthermore, the proportion of genes that could be assigned functional annotation increased, including an increase in the number of major capsid proteins that could be identified. We propose that automatic prediction of stop codon reassignment before annotation is beneficial to downstream viral genomic and metagenomic analyses.
| S-EPMC10769273 | biostudies-literature
Project description:In the mitochondrial genome of the hemichordate Balanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.
| S-EPMC19900 | biostudies-literature
Project description:Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.
| S-EPMC5424465 | biostudies-literature
Project description:Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151). However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability.
| S-EPMC3473164 | biostudies-literature