Nonsense Codon Suppression Research Articles

Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells

The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from the archaeon Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the G1 PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation of two lysine analogs with high selectivity and efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G protein-coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells.

Engineering Translation Components Improve Incorporation of Exotic Amino Acids.

Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.

Future of the Genetic Code.

The methods for establishing synthetic lifeforms with rewritten genetic codes comprising non-canonical amino acids (NCAA) in addition to canonical amino acids (CAA) include proteome-wide replacement of CAA, insertion through suppression of nonsense codon, and insertion via the pyrrolysine and selenocysteine pathways. Proteome-wide reassignments of nonsense codons and sense codons are also under development. These methods enable the application of NCAAs to enrich both fundamental and applied aspects of protein chemistry and biology. Sense codon reassignment to NCAA could incur problems arising from the usage of anticodons as identity elements on tRNA, and possible misreading of NNY codons by UNN anticodons. Evidence suggests that the problem of anticodons as identity elements can be diminished or resolved through removal from the tRNA of all identity elements besides the anticodon, and the problem of misreading of NNY codons by UNN anticodon can be resolved by the retirement of both the UNN anticodon and its complementary NNA codon from the proteome in the event that a restrictive post-transcriptional modification of the UNN anticodon by host enzymes to prevent the misreading cannot be obtained.

Synthetic biology approach for the development of conditionally replicating HIV-1 vaccine.

While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live-attenuated vaccines (LAVs) provided excellent protection in a non-human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV-1 LAVs through controlling HIV-1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid-mediated suppression of amber nonsense codon to generate conditionally replicating HIV-1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies.

Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3).

In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI (+)] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.

Evolved sequence contexts for highly efficient amber suppression with noncanonical amino acids.

The expansion of the genetic code with noncanonical amino acids (ncAA) enables the function of proteins to be tailored with high molecular precision. In this approach, the ncAA is charged to an orthogonal nonsense suppressor tRNA by an aminoacyl-tRNA-synthetase (aaRS) and incorporated into the target protein in vivo by suppression of nonsense codons in the mRNA during ribosomal translation. Compared to sense codon translation, this process occurs with reduced efficiency. However, it is still poorly understood, how the local sequence context of the nonsense codon affects suppression efficiency. Here, we report sequence contexts for highly efficient suppression of the widely used amber codon in E. coli for the orthogonal Methanocaldococcus jannaschii tRNA(Tyr)/TyrRS and Methanosarcina mazei tRNA(Pyl)/PylRS pairs. In vivo selections of sequence context libraries consisting of each two random codons directly up- and downstream of an amber codon afforded contexts with strong preferences for particular mRNA nucleotides and/or amino acids that markedly differed from preferences of contexts obtained in control selections with sense codons. The contexts provided high amber suppression efficiencies with little ncAA-dependence that were transferrable between proteins and resulted in protein expression levels of 70-110% compared to levels of control proteins without amber codon. These sequence contexts represent stable tags for robust and highly efficient incorporation of ncAA into proteins in standard E. coli strains and provide general design rules for the engineering of amber codons into target genes.

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

The reversible Y-O•/Y-OH redox properties of the α3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0±0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°'(Y-O•/Y-OH) of 1026±4 mV versus the normal hydrogen electrode (pH 5.70±0.02) and shows that the fluoro substitutions lower the E°' by -30±3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

Preparation of Translationally Competent tRNA by Direct Chemical Acylation

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

Unnatural amino acids: better than the real things?

Considerable effort has been dedicated to the development of technology for the site-specific incorporation of unnatural amino acids into proteins, with nonsense codon suppression and expressed protein ligation emerging as two of the most promising methods. Recent research advances in which these methods have been applied to study protein function and mechanism are briefly highlighted, and the potential of the methods for efficient, widespread future use in vitro and in vivo is critically evaluated.

Emerging genetic therapies to treat Duchenne muscular dystrophy

Duchenne muscular dystrophy is a progressive muscle degenerative disease caused by dystrophin mutations. The purpose of this review is to highlight two emerging therapies designed to repair the primary genetic defect, called 'exon skipping' and 'nonsense codon suppression'. A drug, PTC124, was identified that suppresses nonsense codon translation termination. PTC124 can lead to restoration of some dystrophin expression in human Duchenne muscular dystrophy muscles with mutations resulting in premature stops. Two drugs developed for exon skipping, PRO051 and AVI-4658, result in the exclusion of exon 51 from mature mRNA. They can restore the translational reading frame to dystrophin transcripts from patients with a particular subset of dystrophin gene deletions and lead to some restoration of dystrophin expression in affected boys' muscle in vivo. Both approaches have concluded phase I trials with no serious adverse events. These novel therapies that act to correct the primary genetic defect of dystrophin deficiency are among the first generation of therapies tailored to correct specific mutations in humans. Thus, they represent paradigm forming approaches to personalized medicine with the potential to lead to life changing treatment for those affected by Duchenne muscular dystrophy.

Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression

High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.

The Effect of Mutation at Thr 295 of Saccharomyces cerevisiae eRF1 on Suppression of Nonsense Codons and eRF1 Structure

The termination of translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1, and eRF3. Two regions in eRF1, at position 281-305 and 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized eRF1 mutants at position 295 from threonine to alanine and serine residues resulting in eRF1(T295A) and eRF1(T295S) respectively. The mutations did not affect the viability or temperature sensitivity of the cells. The stop codons readthrough of the mutants were analyzed in vivo using PGK -stop codon- LACZ gene fusion and the results showed that the suppression of the mutants was increased in all of the codon terminations. The suppression of the UAG codon was the high for both mutants, with a 7-fold increased for eRF1(T295A) and a 9 fold increase for eRF1(T295S). The suppressor activity of eRF1(T295S) was higher compared to that of eRF1(T295A), suggesting that the accuracy of translational termination in eRF1(T295S) was lower than that of eRF1(T295A). Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1 mutants has no significant difference with the wild type. However, substitution of threonine to serine on eRF1(T295S) triggered a secondary structure change on the other motif of the C-terminal domain of eRF1. This observation did not occur for on eRF1(T295A). This suggests that the high stop codon suppression on eRF1(T295S) is probably due to the slight modification of the structure of the C terminal motif.

A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI+] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

The yeast prion [PSI+] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI+] propagation, however the structure-and-function relationship of Hsp104 for [PSI+] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI+] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI+] propagation. However, nine mutants were impaired exclusively for [PSI+], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI+] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI+] prion aggregates and that the lateral channel plays a role specific to [PSI+] prion propagation.

Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast

Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs.

Caged Phosphoproteins

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.

Yeast as a sensor of factors affecting the accuracy of protein synthesis.

The cell monitors and maintains the fidelity of translation during the three stages of protein synthesis: initiation, elongation and termination. Errors can arise by multiple mechanisms, such as altered start site selection, reading frame shifts, misincorporation or nonsense codon suppression. All of these events produce incorrect protein products. Translational accuracy is affected by both cis- and trans-acting elements that insure the proper peptide is synthesized by the protein synthetic machinery. Many cellular components are involved in the accuracy of translation, including RNAs (transfer RNAs, messenger RNAs and ribosomal RNAs) and proteins (ribosomal proteins and translation factors). The yeast Saccharomyces cerevisiae has proven an ideal system to study translational fidelity by integrating genetic approaches with biochemical analysis. This review focuses on the ways studies in yeast have contributed to our understanding of the roles translation factors and the ribosome play in assuring the accuracy of protein synthesis.

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of beta-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA(Ser) gene and the GUS reporter gene resulted in approximately 10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA(Tyr) to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA(Tyr) and tRNA(Met) were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery.

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.