Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids.

In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8-4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15-150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins.

Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein.

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens.

Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5'-phosphorylated and 5'-hydroxylated single-stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double-stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago-based DNA detection toolbox.

A Genetic Study of Nif-Associated Genes in a Hyperthermophilic Methanogen.

Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH, nifD, nifK, and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN, essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCEMethanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

Comprehensive predictions of secondary structures for comparative analysis in different species.

Protein structures are directly linked to biological functions. However, there is a gap of knowledge between the decoded genome and the structure. To bridge the gap, we focused on the secondary structure (SS). From a comprehensive analysis of predicted SS of proteins in different types of organisms, we have arrived at the following findings: The proportions of SS in genomes were different among phylogenic domains. The distributions of strand lengths indicated structural limitations in all of the species. Different from bacteria and archaea, eukaryotes have an abundance of α-helical and random coil proteins. Interestingly, there was a relationship between SS and post-translational modifications. By calculating hydrophobicity moments of helices and strands, highly amphipathic fragments of SS were found, which might be related to the biological functions. In conclusion, comprehensive predictions of SS will provide valuable perspectives to understand the entire protein structures in genomes and will help one to discover or design functional proteins.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

Enhanced production of Dopa-incorporated mussel adhesive protein using engineered translational machineries.

Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows for production of Dopa-incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). To use the Dopa-specific Methanococcus jannaschii TyrRS (MjTyrRS-Dopa), we altered the anticodon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA ) to recognize a tyrosine codon (AUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-incorporated MAP foot protein type 3 (dfp-3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp-3. Even with coexpression of Dopa-recognizing TyrRSs, dfp-3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.

Structure-affinity insights into the Na+ and Ca2+ interactions with multiple sites of a sodium-calcium exchanger.

Selective recognition and transport of Na+ and Ca2+ ions by sodium-calcium exchanger (NCX) proteins is a primary prerequisite for Ca2+ signaling and homeostasis. Twelve ion-coordinating residues are highly conserved among NCXs, and distinct NCX orthologs contain two or three carboxylates, while sharing a common ion-exchange stoichiometry (3Na+ :1Ca2+ ). How these structural differences affect the ion-binding affinity, selectivity, and transport rates remains unclear. Here, the mutational effects of three carboxylates (E54, E213, and D240) were analyzed on the ion-exchange rates in the archaeal NCX from Methanococcus jannaschii and ion-induced structure-affinity changes were monitored by attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR). The D240N mutation elevated the ion-transport rates by twofold to threefold, meaning that the deprotonation of D240 is not essential for transport catalysis. In contrast, mutating E54 or E213 to A, D, N, or Q dramatically decreased the ion-transport rates. ATR-FTIR revealed high- and low-affinity binding of Na+ or Ca2+ with E54 and E213, but not with D240. These findings reveal distinct structure-affinity states at specific ion-binding sites in the inward-facing (IF) and outward-facing orientation. Collectively, two multidentate carboxylate counterparts (E54 and E213) play a critical role in determining the ion coordination/transport in prokaryotic and eukaryotic NCXs, whereas the ortholog substitutions in prokaryotes (aspartate) and eukaryotes (asparagine) at the 240 position affect the ion-transport rates differently (kcat ), probably due to the structural differences in the transition state.

Growth Kinetics, Carbon Isotope Fractionation, and Gene Expression in the Hyperthermophile Methanocaldococcus jannaschii during Hydrogen-Limited Growth and Interspecies Hydrogen Transfer.

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 µM) and low (15 to 27 µM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats.IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Crystal structure of a domain-swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway.

Photoactivatable fluorescent proteins (PA-FPs) are a powerful non-invasive tool in high-resolution live-cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA-FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA-FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl-tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time-resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho-nitrobenzyl-tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7 Å resolution) reveals a dimeric domain-swapped arrangement of sfGFP66ONBY with residues 1-142 of one molecule associating with residues 148-234 from another molecule. This unusual domain-swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

HMMCAS: A Web Tool for the Identification and Domain Annotations of CAS Proteins.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immune systems are discovered in many bacteria and most archaea. These systems are encoded by cas (CRISPR-associated) operons that have an extremely diverse architecture. The most crucial step in the depiction of cas operons composition is the identification of cas genes or Cas proteins. With the continuous increase of the newly sequenced archaeal and bacterial genomes, the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here, we describe HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences. HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast, interactive service based on a comprehensive collection of hidden Markov models of Cas protein family. It can accurately identify the Cas proteins including those fusion proteins, for example the Cas1-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B (Cab. thermophilum B). HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.

Archaeal Unfoldase Counteracts Protein Misfolding Retinopathy in Mice.

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic Archaea, which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ1 knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins.SIGNIFICANCE STATEMENT This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology.

Promiscuity of methionine salvage pathway enzymes in Methanocaldococcus jannaschii.

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

Directed Evolution of Heterologous tRNAs Leads to Reduced Dependence on Post-transcriptional Modifications.

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii ( Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map ms2i6A37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNAOptCUA), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery.

Structural Basis for the Specific Cotranslational Incorporation of p-Boronophenylalanine into Biosynthetic Proteins.

The site-specific incorporation of the non-natural amino acid p-boronophenylalanine (Bpa) into recombinant proteins enables the development of novel carbohydrate-binding functions as well as bioorthogonal chemical modification. To this end, Bpa is genetically encoded by an amber stop codon and cotranslationally inserted into the recombinant polypeptide chain at the ribosome by means of an artificial aminoacyl-tRNA synthetase (aaRS) in combination with a compatible suppressor tRNA. We describe the crystal structure of an aaRS specific for Bpa, which had been engineered on the basis of the TyrRS from Methanocaldococcus jannaschii, in complex with both Bpa and AMP. The substrates are bound in an orientation resembling the aminoacyl-AMP mixed anhydride intermediate and engaged in a network of four hydrogen bonds that allows specific recognition of the boronate moiety by the aaRS. The key determinant of this interaction is the coplanar alignment of its Glu162 carboxylate group with Bpa, which results in a double hydrogen bond with the boronic acid substituent. Our structural study elucidates how a small set of five side chain exchanges within the TyrRS active site can switch its substrate specificity to the hydrophilic amino acid Bpa, thus stimulating the reprogramming of other aaRS to recruit useful non-natural amino acids for next-generation protein engineering.

Engineering posttranslational proofreading to discriminate nonstandard amino acids.

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.