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High-fidelity translation was considered a requirement for living cells. The frozen accident theory suggested that any deviation from the standard genetic code should result in the production of so much mis-made and non-functional proteins that cells cannot remain viable. Studies in bacterial, yeast, and mammalian cells show that significant levels of mistranslation (1-10% per codon) can be tolerated or even beneficial under conditions of oxidative stress. Single tRNA mutants, which occur naturally in the human population, can lead to amino acid mis-incorporation at a codon or set of codons. The rate or level of mistranslation can be difficult or impossible to measure in live cells. We developed a novel red fluorescent protein reporter that is sensitive to serine (Ser) mis-incorporation at proline (Pro) codons. The mCherry Ser151Pro mutant is efficiently produced in

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNA Ser variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNA Gly variants (tRNA Gly CCC , tRNA Gly GCC ) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNA Ala AGC G35C (tRNA Ala ACC ) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNA Gly and tRNA Ala variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNA Gly GCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNA Gly GCC A3G and tRNA Ala AGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.

In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of Huntington's disease-causing alleles. We characterized a tRNAPro mutant that mistranslates proline codons with alanine, and tRNASer mutants, including a tRNASerAGA G35A variant with a phenylalanine anticodon (tRNASerAAA) found in ∼2% of the population. The tRNAPro mutant caused synthetic toxicity with a deleterious huntingtin poly-glutamine (polyQ) allele in neuronal cells. The tRNASerAAA variant showed synthetic toxicity with proteasome inhibition but did not enhance toxicity of the huntingtin allele. Cells mistranslating phenylalanine or proline codons with serine had significantly reduced rates of protein synthesis. Mistranslating cells were slow but effective in forming insoluble polyQ aggregates, defective in protein and aggregate degradation, and resistant to the neuroprotective integrated stress response inhibitor (ISRIB). Our findings identify mistranslating tRNA variants as genetic factors that slow protein aggregation kinetics, inhibit aggregate clearance, and increase drug resistance in cellular models of neurodegenerative disease., Graphical Abstract Graphical AbstractA human tRNASer variant found in 1.8% of the population directs serine mis-incorporation and inhibits protein synthesis. Mistranslating cells were drug-resistant and defective in poly-glutamine aggregate clearance in models of Huntington's disease.

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼10(11) peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1(T308), the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

Ubiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl-lysine addition to minimize mistranslated proteins and maximize site-specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl-lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns.

High-fidelity protein synthesis requires properly aminoacylated transfer RNAs (tRNAs), yet diverse cell types, from bacteria to humans, show a surprising ability to tolerate errors in translation resulting from mutations in tRNAs, aminoacyl-tRNA synthetases, and other components of protein synthesis. Recently, we characterized a tRNASerAGA G35A mutant (tRNASerAAA) that occurs in 2% of the human population. The mutant tRNA decodes phenylalanine codons with serine, inhibits protein synthesis, and is defective in protein and aggregate degradation. Here, we used cell culture models to test our hypothesis that tRNA-dependent mistranslation will exacerbate toxicity caused by amyotrophic lateral sclerosis (ALS)-associated protein aggregation. Relative to wild-type tRNA, we found cells expressing tRNASerAAA showed slower but effective aggregation of the fused in sarcoma (FUS) protein. Despite reduced levels in mistranslating cells, wild-type FUS aggregates showed similar toxicity in mistranslating cells and normal cells. The aggregation kinetics of the ALS-causative FUS R521C variant were distinct and more toxic in mistranslating cells, where rapid FUS aggregation caused cells to rupture. We observed synthetic toxicity in neuroblastoma cells co-expressing the mistranslating tRNA mutant and the ALS-causative FUS R521C variant. Our data demonstrate that a naturally occurring human tRNA variant enhances cellular toxicity associated with a known causative allele for neurodegenerative disease.

The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutumHASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90–95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.

Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNAHis). Mutations in HARS cause the human genetic disorders Usher syndrome type 3B (USH3B) and Charcot-Marie-Tooth syndrome type 2W (CMT2W). Treatment for these diseases remains symptomatic, and no disease specific treatments are currently available. Mutations in HARS can lead to destabilization of the enzyme, reduced aminoacylation, and decreased histidine incorporation into the proteome. Other mutations lead to a toxic gain-of-function and mistranslation of non-cognate amino acids in response to histidine codons, which can be rescued by histidine supplementation in vitro. We discuss recent advances in characterizing HARS mutations and potential applications of amino acid and tRNA therapy for future gene and allele specific therapy.

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNA

Transfer RNAs are required to translate genetic information into proteins as well as regulate other cellular processes. Nucleotide changes in tRNAs can result in loss or gain of function that impact the composition and fidelity of the proteome. Despite links between tRNA variation and disease, the importance of cytoplasmic tRNA variation has been overlooked. Using a custom capture panel, we sequenced 605 human tRNA-encoding genes from 84 individuals. We developed a bioinformatic pipeline that allows more accurate tRNA read mapping and identifies multiple polymorphisms occurring within the same variant. Our analysis identified 522 unique tRNA-encoding sequences that differed from the reference genome from 84 individuals. Each individual had ~66 tRNA variants including nine variants found in less than 5% of our sample group. Variants were identified throughout the tRNA structure with 17% predicted to enhance function. Eighteen anticodon mutants were identified including potentially mistranslating tRNAs; e.g., a tRNASer that decodes Phe codons. Similar engineered tRNA variants were previously shown to inhibit cell growth, increase apoptosis and induce the unfolded protein response in mammalian cell cultures and chick embryos. Our analysis shows that human tRNA variation has been underestimated. We conclude that the large number of tRNA genes provides a buffer enabling the emergence of variants, some of which could contribute to disease.

Perfectly accurate translation of mRNA into protein is not a prerequisite for life. Resulting from errors in protein synthesis, mistranslation occurs in all cells, including human cells. The human genome encodes >600 tRNA genes, providing both the raw material for genetic variation and a buffer to ensure that resulting translation errors occur at tolerable levels. On the basis of data from the 1000 Genomes Project, we highlight the unanticipated prevalence of mistranslating tRNA variants in the human population and review studies on synthetic and natural tRNA mutations that cause mistranslation or de-regulate protein synthesis. Although mitochondrial tRNA variants are well known to drive human diseases, including developmental disorders, few studies have revealed a role for human cytoplasmic tRNA mutants in disease. In the context of the unexpectedly large number of tRNA variants in the human population, the emerging literature suggests that human diseases may be affected by natural tRNA variants that cause mistranslation or de-regulate tRNA expression and nucleotide modification. This review highlights examples relevant to genetic disorders, cancer, and neurodegeneration in which cytoplasmic tRNA variants directly cause or exacerbate disease and disease-linked phenotypes in cells, animal models, and humans. In the near future, tRNAs may be recognized as useful genetic markers to predict the onset or severity of human disease.

Protein kinase B (AKT1) is hyper-activated in diverse human tumors. AKT1 is activated by phosphorylation at two key regulatory sites, Thr308 and Ser473. Active AKT1 phosphorylates many, perhaps hundreds, of downstream cellular targets in the cytosol and nucleus. AKT1 is well-known for phosphorylating proteins that regulate cell survival and apoptosis, however, the full catalog of AKT1 substrates remains unknown. Using peptide arrays, we recently discovered that each phosphorylated form of AKT1 (pAKT1S473, pAKT1T308, and ppAKT1S473,T308) has a distinct substrate specificity, and these data were used to predict potential new AKT1 substrates. To test the high-confidence predictions, we synthesized target peptides representing putative AKT1 substrates. Peptides substrates were synthesized by solid phase synthesis and their purity was confirmed by mass spectrometry. Most of the predicted peptides showed phosphate accepting activity similar to or greater than that observed with a peptide derived from a well-established AKT1 substrate, glycogen synthase kinase 3β (GSK-3β). Among the novel substrates, AKT1 was most active with peptides representing PIP3-binding protein Rab11 family-interacting protein 2 and cysteinyl leukotriene receptor 1, indicating their potential role in AKT1-dependent cellular signaling. The ppAKT1S473,T308 enzyme was highly selective for peptides containing a patch of basic residues at −5, −4, −3 and aromatic residues (Phe/Tyr) at +1 positions from the phosphorylation site. The pAKT1S473 variant preferred more acidic peptides, Ser or Pro at +4, and was agnostic to the residue at −5. The data further support our hypothesis that Ser473 phosphorylation plays a key role in modulating AKT1 substrate selectivity.

Transfer RNA (tRNA) has a long-established role in protein synthesis. The tRNA molecule serves as an adaptor [1] between the genetic instructions written in nucleic acid sequences and the protein p...

Expanding the genetic code using non-canonical amino acids has revolutionized synthetic biology. In a recent issue of Cell Chemical Biology, Yanagisawa et al. (2019) expanded our knowledge of orthogonal translation systems by presenting a tour de force structural characterization of multiple genetic code expansion systems based on pyrrolysyl-tRNA synthetase.

It is increasingly apparent that the genetic code is not static and that organisms use variations in the genetic code for selective advantage. Berg..., The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele (tti2-L187P) in Saccharomyces cerevisiae to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNASer containing a proline anticodon (UGG) is toxic, using our selection system we identified four tRNASer UGG variants, each with a single mutation, that mistranslate at a tolerable level. Mistranslating tRNALeu UGG variants were also obtained, demonstrating the generality of the approach. We characterized two of the tRNASer UGG variants. One contained a G26A mutation, which reduced cell growth to 70% of the wild-type rate, induced a heat shock response, and was lost in the absence of selection. The reduced toxicity of tRNASer UGG-G26A is likely through increased turnover of the tRNA, as lack of methylation at G26 leads to degradation via the rapid tRNA decay pathway. The second tRNASer UGG variant, with a G9A mutation, had minimal effect on cell growth, was relatively stable in cells, and gave rise to less of a heat shock response. In vitro, the G9A mutation decreases aminoacylation and affects folding of the tRNA. Notably, the G26A and G9A mutations were phenotypically neutral in the context of an otherwise wild-type tRNASer. These experiments reveal a model for genetic code evolution in which tRNA anticodon mutations and mistranslation evolve through phenotypically ambivalent intermediates that reduce tRNA function.

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

Despite the general requirement for translation fidelity, mistranslation can be an adaptive response. We selected spontaneous second site mutations that suppress the stress sensitivity caused by a Saccharomyces cerevisiae tti2 allele with a Leu to Pro mutation at residue 187, identifying a single nucleotide mutation at the same position (C70U) in four tRNAProUGG genes. Linkage analysis and suppression by SUF9G3:U70 expressed from a centromeric plasmid confirmed the causative nature of the suppressor mutation. Since the mutation incorporates the G3:U70 identity element for alanyl-tRNA synthetase into tRNAPro, we hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. A strain expressing Tti2L187A was not stress sensitive. In vitro, tRNAProUGG (C70U) was mis-aminoacylated with alanine by alanyl–tRNA synthetase, but was not a substrate for prolyl–tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ∼6%. Mistranslating cells expressing SUF9G3:U70 induce a partial heat shock response but grow nearly identically to wild-type. Introducing the same G3:U70 mutation in SUF2 (tRNAProAGG) suppressed a second tti2 allele (tti2L50P). We have thus identified a strategy that allows mistranslation to suppress deleterious missense Pro mutations in Tti2.

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308, phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.

Aims: Thioredoxin reductase 1 (TrxR1) is a cancer target and essential selenoprotein that defends the cell against reactive oxygen species and regulates cellular signaling and redox pathways. Previous cell-based studies correlated TrxR1 acetylation with modulated cellular reduction activity, yet the function of specific acetylation sites on TrxR1 remains unknown. Innovation: We produced site-specifically acetylated TrxR1 variants that also contain selenocysteine (Sec). We demonstrated efficient high-fidelity protein synthesis with 22 different amino acids by simultaneous UAG codon reassignment to Nɛ-acetyl-lysine and UGA codon recoding to Sec. Results: We characterized TrxR1 variants acetylated at physiologically relevant sites and found that single acetylation sites increased TrxR1 activity, enhancing the apparent catalytic rate up to 2.7-fold. The activity increase in acetylated TrxR1 (acTrxR1) is reversible and is reduced following deacetylation with histone deacetylase. Conclusion: Here we present a novel mechanism through which acetylation increases TrxR1 activity by destabilizing low-activity TrxR1 multimers, increasing the population of active dimeric TrxR1. Antioxid. Redox Signal. 29, 377–388.

Malfunction of the ubiquitin (Ub) E3 ligase, parkin, leads to defects in mitophagy and protein quality control linked to Parkinson's disease. Parkin activity is stimulated by phosphorylation of Ub at Ser65 (pUbS65). Since the upstream kinase is only known for Ser65 (PINK1), the biochemical function of other phosphorylation sites on Ub remain largely unknown. We used fluorescently labelled and site-specifically phosphorylated Ub substrates to quantitatively relate the position and stoichiometry of Ub phosphorylation to parkin activation. Fluorescence measurements show that pUbS65-stimulated parkin is 5-fold more active than auto-inhibited and un-stimulated parkin, which catalyzes a basal level of auto-ubiquitination. We consistently observed a low but detectable level of parkin activity with pUbS12. Strikingly, pUbS57 hyper-activates parkin, and our data demonstrate that parkin is able to selectively synthesize poly-pUbS57 chains, even when 90% of the Ub in the reaction is un-phosphorylated. We further found that parkin ubiquitinates its physiological substrate Miro-1 with chains solely composed of pUbS65 and more efficiently with pUbS57 chains. Parkin hyper-activation by pUbS57 demonstrates the first PINK1-independent route to active parkin, revealing the roles of multiple ubiquitin phosphorylation sites in governing parkin stimulation and catalytic activity. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.

Der Selenocystein(Sec)-Einbau in Proteine erfolgt in der Natur durch die kotranslationale Umkodierung eines UGA-Stopp-Codons. Diese Studie zeigt nun, dass Sec nicht ausschlieslich durch UGA, sondern vielmehr durch 58 aller 64 moglichen Codons kodiert werden kann. Hierbei erlauben 15 Codons nicht nur die vollstandige Umkodierung von ihrer ursprunglichen Bedeutung als kanonische Aminosaure zu Selenocystein, sondern sie fuhren zu Proteinausbeuten, die um mehr als das Zehnfache gesteigert sind. Der hoch effiziente Mechanismus zur Selenocystein-Rekodierung wird anhand zweier Reporterenzyme, der Escherichia-coli-Formiatdehydrogenase und der humanen Thioredoxinreduktase, beschrieben. Da der Selenocysteineinbau an der Position eines UGA-Stopp-Codons zwangslaufig mit translationaler Termination konkurriert, war es umso erstaunlicher, dass die Selenocystein-Einbaumaschinerie wahrend der Umkodierung von Sense-Codons erfolgreich mit den im Uberfluss vorhandenen regularen Aminoacyl-tRNAs konkurriert.

Genetic code expansion for synthesis of proteins containing noncanonical amino acids is a rapidly growing field in synthetic biology. Creating optimal orthogonal translation systems will require re-engineering central components of the protein synthesis machinery on the basis of a solid mechanistic biochemical understanding of the synthetic process.

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10−4 in yeast) with a limited impact on phenotype. Previously, we selected a tRNAPro containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNAPro (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 4–6% in HEK 293T cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.

Background The conservation of the genetic code indicates that there was a single origin, but like all genetic material, the cell's interpretation of the code is subject to evolutionary pressure. Single nucleotide variations in tRNA sequences can modulate codon assignments by altering codon-anticodon pairing or tRNA charging. Either can increase translation errors and even change the code. The frozen accident hypothesis argued that changes to the code would destabilize the proteome and reduce fitness. In studies of model organisms, mistranslation often acts as an adaptive response. These studies reveal evolutionary conserved mechanisms to maintain proteostasis even during high rates of mistranslation. Scope of review This review discusses the evolutionary basis of altered genetic codes, how mistranslation is identified, and how deviations to the genetic code are exploited. We revisit early discoveries of genetic code deviations and provide examples of adaptive mistranslation events in nature. Lastly, we highlight innovations in synthetic biology to expand the genetic code. Major conclusions The genetic code is still evolving. Mistranslation increases proteomic diversity that enables cells to survive stress conditions or suppress a deleterious allele. Genetic code variants have been identified by genome and metagenome sequence analyses, suppressor genetics, and biochemical characterization. General significance Understanding the mechanisms of translation and genetic code deviations enables the design of new codes to produce novel proteins. Engineering the translation machinery and expanding the genetic code to incorporate non-canonical amino acids are valuable tools in synthetic biology that are impacting biomedical research. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue.

The activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb(S65) ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (ΔRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb(S20) failed to stimulate parkin, parkin was partially active with pUb(S12) . We observed significant ubiquitination when pUb(S65) was the sole substrate.

Histidine transfer RNA (tRNA) is unique among tRNA species as it carries an additional nucleotide at its 5' terminus. This unusual G(-1) residue is the major tRNA(His) identity element, and essential for recognition by the cognate histidyl-tRNA synthetase to allow efficient His-tRNA(His) formation. In many organisms G(-1) is added post-transcriptionally as part of the tRNA maturation process. tRNA(His) guanylyltransferase (Thg1) specifically adds the guanylyate residue by recognizing the tRNA(His) anticodon. Thg1 homologs from all three domains of life have been the subject of exciting research that gave rise to a detailed biochemical, structural and phylogenetic enzyme characterization. Thg1 homologs are phylogenetically classified into eukaryal- and archaeal-type enzymes differing characteristically in their cofactor requirements and specificity. Yeast Thg1 displays a unique but limited ability to add 2-3 G or C residues to mutant tRNA substrates, thus catalyzing a 3' → 5' RNA polymerization. Archaeal-type Thg1, which has been horizontally transferred to certain bacteria and few eukarya, displays a more relaxed substrate range and may play additional roles in tRNA editing and repair. The crystal structure of human Thg1 revealed a fascinating structural similarity to 5' → 3' polymerases, indicating that Thg1 derives from classical polymerases and evolved to assume its specific function in tRNA(His) processing.