tRNA Pair Research Articles

Expanded Cellular Amino Acid Pools Containing Phosphoserine, Phosphothreonine, and Phosphotyrosine

Adding nonstandard amino acids to the genetic code of E. coli expands the chemical and biological functional space for proteins. This is accomplished with engineered, orthogonal aminoacyl-tRNA synthetase and tRNA pairs that require a nonstandard amino acid in sufficient intracellular quantities to support protein synthesis. While cotranslational insertion of phosphoserine into proteins has been accomplished, conditions that modulate intracellular phosphoamino acid concentrations are still poorly understood. Here we used genetic and metabolic engineering to increase the free intracellular levels of phosphoserine in E. coli. We show that deletion of the phosphoserine phosphatase serB elevates the intracellular levels of phosphoserine within ranges comparable to those of standard amino acids. These new conditions improved insertion of phosphoserine into recombinant proteins. Surprisingly, we also observed dramatic increases in intracellular levels of phosphothreonine and phosphotyrosine when WT cells were grown in LB with supplemented phosphothreonine and serB deficient cells were grown in low phosphate media with supplemented phosphotyrosine, respectively. These findings remove a major barrier for further expansion of the genetic code with additional phosphorylated amino acids.

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

EF-G:A-Site Trna Distance Changes during Translocation

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Attaching a fluorescent probe within domain IV of EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. Two different sites on L11, position 38 or position 87, as well as two different A-site tRNAs, Phe-tRNAPhe or Val-tRNAVal were labeled with Cy5. The EF-G:L11 FRET efficiency is constant during EF-G occupancy of the ribosome (300 - 400 ms), whereas the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. For both tRNAs, the EF-G:tRNA pair spends most of its time in the intermediate FRET state, with shorter lifetimes in the high and low FRET states. Each transition in the FRET value corresponds to a relative distance change of about 5 to 7 A, so in total, we capture a 12 A relative movement of A-site tRNA away from EF-G during translocation. EF-G:A-site tRNA FRET is sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state. In addition, the translocation inhibitors viomycin and spectinomycin block formation of the low FRET state, although spectinomycin allows more movement between EF-G and tRNA than viomycin. Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNAHis have distinctive identity elements, we constructed E. coli tRNAHis CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNAHis CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNAHis CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNAHis CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.

Directed evolution of genetic parts and circuits by compartmentalized partnered replication

Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.

Transfer RNA Misidentification Scrambles Sense Codon Recoding

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(Pyl)(CCG)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(Pyl)(CCG) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(Pyl)(CCG), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.

Aminoacylating Urzymes Challenge the RNA World Hypothesis

We describe experimental evidence that ancestral peptide catalysts substantially accelerated development of genetic coding. Structurally invariant 120-130-residue Urzymes (Ur = primitive plus enzyme) derived from Class I and Class II aminoacyl-tRNA synthetases (aaRSs) acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated amino acids. These new data allow us to demonstrate statistically indistinguishable catalytic profiles for Class I and II aaRSs in both amino acid activation and tRNA acylation, over a time period extending to well before the assembly of full-length enzymes and even further before the Last Universal Common Ancestor. Both Urzymes also exhibit ∼60% of the contemporary catalytic proficiencies. Moreover, they are linked by ancestral sense/antisense genetic coding, and their evident modularities suggest descent from even simpler ancestral pairs also coded by opposite strands of the same gene. Thus, aaRS Urzymes substantially pre-date modern aaRS but are, nevertheless, highly evolved. Their unexpectedly advanced catalytic repertoires, sense/antisense coding, and ancestral modularities imply considerable prior protein-tRNA co-evolution. Further, unlike ribozymes that motivated the RNA World hypothesis, Class I and II Urzyme·tRNA pairs represent consensus ancestral forms sufficient for codon-directed synthesis of nonrandom peptides. By tracing aaRS catalytic activities back to simpler ancestral peptides, we demonstrate key steps for a simpler and hence more probable peptide·RNA development of rapid coding systems matching amino acids with anticodon trinucleotides.

Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNASec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNASec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNASec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNASecs, A. aeolicus tRNASec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNASec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNASec. Similar interactions exist in the reported tRNASer and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNASec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNASec to enter the SerRS catalytic site.

Haste Makes Waste

Cell Biology mTORC1 is a protein kinase complex that regulates many biological processes, including cell growth and proliferation, and that has a primary role in the control of protein synthesis. Inhibition of mTORC1 increases life span, and partial inhibition of translation mimics the effect on life span, indicating that effects of mTORC1 on life span might be related to effects on protein synthesis. Conn and Qian present a mechanism by which reduced rates of protein synthesis might extend life span. They found essentially that haste makes waste. When mTORC1 activity was high and cells synthesized polypeptides rapidly, the stability of a newly synthesized fluorescent reporter protein was diminished. Inhibition of mTORC1, on the other hand, slowed protein synthesis through effects on translation initiation and elongation, and improved the fidelity of the process. Slower translation may allow more time for correct tRNA pairing or for cotranslational processes that promote proper folding and may thus result in fewer misincorporated amino acids or misfolded proteins, either of which would tend to reduce protein stability. Sci. Signal. 6 , ra24 (2013).

A Tryptophanyl‐tRNA Synthetase/tRNA Pair for Unnatural Amino Acid Mutagenesis in E. coli

Selected: A tryptophanyl-tRNA synthetase(TrpRS)/tRNA pair was evolved to genetically encode tryptophan analogues and other unnatural amino acids with large side chains in E. coli. A selection scheme was employed to identify TrpRS variants able to selectively charge tRNACUATrp with Trp analogues (see picture). Substitution of Trp66 in enhanced cyan fluorescent protein (ECFP) with these Trp analogues afforded ECFP variants with novel spectral properties. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

For novel insights into the pathogenicity of Candida albicans, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p-azido-L-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyl-tRNA synthetase and a suppressor tRNA pair (EcTyrtRNA(CUA)) derived from Escherichia coli, which was previously reported to be orthogonal in Saccharomyces cerevisiae. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and EcTyrtRNA(CUA) for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecular-weight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

Expanding the Genetic Code in Xenopus laevis Oocytes

Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.

Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair

Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichiacoli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e. proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kcat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4×10−5s−1. Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported cell-free accumulation of two different modified proteins (450±20μg/ml CAT109pAzF and 428±2μg/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP cell-free recipe.

Photoinactivation of Glutamate Receptors using a Genetically Encoded Unnatural Amino Acid

Site-specific incorporation of unnatural amino acids has wide-ranging applications in the structure-function studies of proteins, offering high specificity, arbitrary environmental sensitivity of incorporation and a nearly unlimited variety of unnatural side chains. We have used photo-activatable unnatural amino acids to probe the activation and desensitization of glutamate receptors (GluRs), ligand-gated ion channels that mediate fast excitatory synaptic transmission in the brain. We introduced the unnatural amino acid cross-linker p-Benzoyl-Phenylalanine (Bpa) to the AMPA receptor GluA2 expressed in mammalian cell lines. Sites of interest were replaced by an amber stop codon (TAG). The stop codon was suppressed using a genetically-encoded exogenous tRNA and tRNA-synthetase pair that incorporate Bpa (Ye et al, 2008 J Biol Chem). We initially chose sites in the lower (D2) lobes of the LBD (S729, G725), which have been previously shown to form intermolecular disulfide cross-links that inhibit the receptor (Armstrong et al, 2006 Cell; Plested and Mayer, 2009 J. Neurosci). Patch clamp electrophysiology coupled to rapid perfusion demonstrated that Bpa-rescued GluRs had similar properties to wild-type GluRs. Receptors could be trapped in inactive conformational states upon UV irradiation and subsequent photo-cross-linking, consistent with previous work. Irreversible trapping was characterized by near-complete peak current reduction (95 %) in less than five seconds cumulative exposure to UV at 365 nm from a mercury lamp. The cross-linking rate depended on UV intensity and exposure time. Wild-type channels were unaffected by similar UV exposures. Our results demonstrate that genetically-encoded unnatural amino acids such as Bpa provide an convenient strategy to control structural rearrangements of ligand-gated ion channels in mammalian cells.

Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms are not evolutionarily conserved, and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error prone, most notably a Mycoplasma example that displayed a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma PheRS was found to lack canonical editing activity, relying instead on discrimination against the non-cognate amino acid by kinetic proofreading. This mechanism of discrimination is inadequate for organisms where translation is more accurate, as Mycoplasma PheRS failed to support Escherichia coli growth. However, minor changes in the defunct editing domain of the Mycoplasma enzyme were sufficient to restore E. coli growth, indicating that translational accuracy is an evolutionarily selectable trait.

Performance Analysis of Orthogonal Pairs Designed for an Expanded Eukaryotic Genetic Code

BackgroundThe suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNACUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid.Methodology/Principal FindingsWe used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs.Conclusions/SignificanceO-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detailed analysis of their catalytic properties is still missing. Our study provides a comprehensive scrutiny of o-pairs developed for the site-specific incorporation of reactive ncAAs in S. cerevisiae. It suggests that future development of o-pairs as efficient biotechnological tools will greatly benefit from sound characterization in vivo and in vitro in parallel to monitoring intracellular ncAA levels.

High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNACUAopt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.

Genetically encoded alkenyl–pyrrolysine analogues for thiol–ene reaction mediated site-specific protein labeling

A series of alkene-bearing pyrrolysine analogues were synthesized and subsequently incorporated into proteins at two sites by a mutant PylRS–tRNA pair with excellent efficiency. This strategy allowed the site-specific labeling of proteins carrying single or double genetically encoded alkene handles via bioorthogonal thiol–ene ligation reactions.

Genetically Encoding Bioorthogonal Functional Groups for Site-selective Protein Labeling

Site-selective protein labeling is an indispensible approach in the currently intense chemical biology research area. Studies involving site-selective protein labeling span from the protein dynamic analysis in vitro to the proteinprotein interaction investigation in living cells. In the past decade, multiple methods have been introduced to achieve site-selective protein labeling. These include genetic fusion of green fluorescent protein and its derivatives, selective chemical labeling of proteins with fusion tags, and site-specific modification of noncanonical amino acids that are genetically encoded. Using evolved orthogonal aminoacyl-tRNA synthetase-nonsense suppressor tRNA pairs, noncanonical amino acids with bioorthogonal functional groups such as azide, alkyne, tetrazine, alkene, keto, phenylhalide, etc. have been genetically incorporated into proteins in E. coli, yeast, and mammalian cells. Genetic encoding of these noncanonical amino acids enables multiple ways for site-selective protein labeling both in vitro and in vivo, allowing diverse strategies to interrogate protein functions. This review intends to provide a brief introduction to the genetic noncanonical amino acid incorporation technique and recent progresses in applying this technique to achieve site-selective protein labeling.