601 results on '"Dieter Söll"'
Search Results
2. Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids
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Han-Kai Jiang, Jui-Hung Weng, Yi-Hui Wang, Jo-Chu Tsou, Pei-Jung Chen, An-Li Andrea Ko, Dieter Söll, Ming-Daw Tsai, and Yane-Shih Wang
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pyrrolysyl-tRNA synthetase ,genetic code expansion ,noncanonical amino acids ,D-phenylalanine analogs ,synthetic biology ,amber suppression ,Genetics ,QH426-470 - Abstract
Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs’ unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase’s (PylRS’) remarkable substrate polyspecificity, this work attempts to increase PylRS’ ability in directly charging tRNAPyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins’ physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.
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- 2023
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3. Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins
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Pearl Morosky, Cody Comyns, Lance G. A. Nunes, Christina Z. Chung, Peter R. Hoffmann, Dieter Söll, Oscar Vargas-Rodriguez, and Natalie Krahn
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selenoproteins ,acetyl-lysine ,post-translational modifications ,genetic code expansion ,selenocysteine ,synthetic biology ,Biology (General) ,QH301-705.5 - Abstract
Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein’s function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of Nε-acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.
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- 2023
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4. Harnessing selenocysteine to enhance microbial cell factories for hydrogen production
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Armaan Patel, David W. Mulder, Dieter Söll, and Natalie Krahn
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Selenocysteine ,cell factories ,biofuel ,allo-tRNA ,Selenoprotein ,C1 microbes ,Chemistry ,QD1-999 - Abstract
Hydrogen is a clean, renewable energy source, that when combined with oxygen, produces heat and electricity with only water vapor as a biproduct. Furthermore, it has the highest energy content by weight of all known fuels. As a result, various strategies have engineered methods to produce hydrogen efficiently and in quantities that are of interest to the economy. To approach the notion of producing hydrogen from a biological perspective, we take our attention to hydrogenases which are naturally produced in microbes. These organisms have the machinery to produce hydrogen, which when cleverly engineered, could be useful in cell factories resulting in large production of hydrogen. Not all hydrogenases are efficient at hydrogen production, and those that are, tend to be oxygen sensitive. Therefore, we provide a new perspective on introducing selenocysteine, a highly reactive proteinogenic amino acid, as a strategy towards engineering hydrogenases with enhanced hydrogen production, or increased oxygen tolerance.
- Published
- 2022
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5. Multiplex suppression of four quadruplet codons via tRNA directed evolution
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Erika A. DeBenedictis, Gavriela D. Carver, Christina Z. Chung, Dieter Söll, and Ahmed H. Badran
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Science - Abstract
Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.
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- 2021
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6. Unconventional genetic code systems in archaea
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Kexin Meng, Christina Z. Chung, Dieter Söll, and Natalie Krahn
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archaea ,selenocysteine ,pyrrolysine ,phosphoserine ,genetic code expansion ,Microbiology ,QR1-502 - Abstract
Archaea constitute the third domain of life, distinct from bacteria and eukaryotes given their ability to tolerate extreme environments. To survive these harsh conditions, certain archaeal lineages possess unique genetic code systems to encode either selenocysteine or pyrrolysine, rare amino acids not found in all organisms. Furthermore, archaea utilize alternate tRNA-dependent pathways to biosynthesize and incorporate members of the 20 canonical amino acids. Recent discoveries of new archaeal species have revealed the co-occurrence of these genetic code systems within a single lineage. This review discusses the diverse genetic code systems of archaea, while detailing the associated biochemical elements and molecular mechanisms.
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- 2022
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7. Diversification of aminoacyl-tRNA synthetase activities via genomic duplication
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Natalie Krahn, Dieter Söll, and Oscar Vargas-Rodriguez
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gene duplication ,aminoacyl-tRNA synthetase ,evolution ,translation ,tRNA ,noncanonical functions ,Physiology ,QP1-981 - Abstract
Intricate evolutionary events enabled the emergence of the full set of aminoacyl-tRNA synthetase (aaRS) families that define the genetic code. The diversification of aaRSs has continued in organisms from all domains of life, yielding aaRSs with unique characteristics as well as aaRS-like proteins with innovative functions outside translation. Recent bioinformatic analyses have revealed the extensive occurrence and phylogenetic diversity of aaRS gene duplication involving every synthetase family. However, only a fraction of these duplicated genes has been characterized, leaving many with biological functions yet to be discovered. Here we discuss how genomic duplication is associated with the occurrence of novel aaRSs and aaRS-like proteins that provide adaptive advantages to their hosts. We illustrate the variety of activities that have evolved from the primordial aaRS catalytic sites. This precedent underscores the need to investigate currently unexplored aaRS genomic duplications as they may hold a key to the discovery of exciting biological processes, new drug targets, important bioactive molecules, and tools for synthetic biology applications.
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- 2022
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8. Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant
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Jonathan T. Fischer, Dieter Söll, and Jeffery M. Tharp
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directed evolution ,PANCE ,PylRS ,noncanonical amino acid ,tRNA ,orthogonal ,Biology (General) ,QH301-705.5 - Abstract
Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS (MaPylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of MaPylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve MaPylRS to efficiently incorporate Nε-Boc-l-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRSopt) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRSopt can be used to enhance previously engineered PylRS constructs such as MaPylRSN166S, and PylRSopt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.
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- 2022
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9. Measuring the tolerance of the genetic code to altered codon size
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Erika Alden DeBenedictis, Dieter Söll, and Kevin M Esvelt
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genetic code expansion ,tRNA ,directed evolution ,quadruplet codon ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet codon, rather than quadruplet codon, genetic code? Here, we investigate the tolerance of the Escherichia coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all 20 canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.
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- 2022
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10. Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems
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Takahito Mukai, Kazuaki Amikura, Xian Fu, Dieter Söll, and Ana Crnković
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aminoacyl-tRNA synthetases ,O-phosphoseryl-tRNA synthetase ,genetic code ,tRNA ,cysteine ,selenocysteine ,Genetics ,QH426-470 - Abstract
Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.
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- 2022
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11. Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro
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Omer Ad, Kyle S. Hoffman, Andrew G. Cairns, Aaron L. Featherston, Scott J. Miller, Dieter Söll, and Alanna Schepartz
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Chemistry ,QD1-999 - Published
- 2019
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12. Naturally Occurring tRNAs With Non-canonical Structures
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Natalie Krahn, Jonathan T. Fischer, and Dieter Söll
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tRNA ,non-canonical ,genetic code expansion ,identity elements ,translation ,selenocysteine ,Microbiology ,QR1-502 - Abstract
Transfer RNA (tRNA) is the central molecule in genetically encoded protein synthesis. Most tRNA species were found to be very similar in structure: the well-known cloverleaf secondary structure and L-shaped tertiary structure. Furthermore, the length of the acceptor arm, T-arm, and anticodon arm were found to be closely conserved. Later research discovered naturally occurring, active tRNAs that did not fit the established ‘canonical’ tRNA structure. This review discusses the non-canonical structures of some well-characterized natural tRNA species and describes how these structures relate to their role in translation. Additionally, we highlight some newly discovered tRNAs in which the structure–function relationship is not yet fully understood.
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- 2020
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13. Using Genetic Code Expansion for Protein Biochemical Studies
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Christina Z. Chung, Kazuaki Amikura, and Dieter Söll
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genetic code expansion ,non-canonical amino acids ,protein labeling ,protein purification ,protein–protein interactions ,Biotechnology ,TP248.13-248.65 - Abstract
Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue- or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein–protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein–protein interactions and offer considerations for future work in expanding genetic code expansion methods.
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- 2020
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14. Crystal structures of the human elongation factor eEFSec suggest a non-canonical mechanism for selenocysteine incorporation
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Malgorzata Dobosz-Bartoszek, Mark H. Pinkerton, Zbyszek Otwinowski, Srinivas Chakravarthy, Dieter Söll, Paul R. Copeland, and Miljan Simonović
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Science - Abstract
Specialized translation elongation factors (eEFSec and SelB) promote selenocysteine incorporation into proteins. Here, the authors report the structure of human eEFSec, examine its interactions with guanine nucleotides, and propose a non-canonical mechanism for decoding selenocysteine.
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- 2016
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15. Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion
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Ana Crnković, Tateki Suzuki, Dieter Söll, and Noah M. Reynolds
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Chemistry ,QD1-999 - Abstract
Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl‐tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS⋅tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS. This work is licensed under a Creative Commons Attribution 4.0 International License.
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- 2016
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16. RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea
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Takahito Mukai, Ana Crnković, Takuya Umehara, Natalia N. Ivanova, Nikos C. Kyrpides, and Dieter Söll
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biochemistry ,bioinformatics ,cysteine biosynthesis ,genetic code ,translation ,Microbiology ,QR1-502 - Abstract
ABSTRACT The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria (“Candidatus Parcubacteria” and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes. IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.
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- 2017
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17. Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids
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Ana Crnković, Oscar Vargas-Rodriguez, Anna Merkuryev, and Dieter Söll
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noncanonical amino acids ,genetic code expansion ,protein translation ,tRNA ,aminoacyl-tRNA synthetases ,posttranscriptional modifications ,phosphoserine ,Technology ,Biology (General) ,QH301-705.5 - Abstract
Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.
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- 2018
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18. Aminoacyl-tRNA Synthesis in Methanogenic Archaea
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Dragana Korenčić, Ivan Ahel, and Dieter Söll
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aminoacyl-tRNA ,methanogenic Archaea ,evolution ,tRNA ,translation ,Biotechnology ,TP248.13-248.65 ,Food processing and manufacture ,TP368-456 - Abstract
Aminoacyl-tRNA synthetases (AARSs) are essential for faithful translation of the genetic code and have long been studied intensively. Major discoveries explained basic principles of how amino acids are paired to their cognate tRNAs to ensure high fidelity of translation. However, advances in genomics instigated identification of novel enzymes and pathways to aminoacyl-tRNA synthesis. In that respect methanogenic Archaea are particularly prominent, most of which possess non-canonical routes to synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Additionally, some methanogenic seryl-tRNA synthetases are only marginally related to their homologues outside the archaeal kingdom, while other AARSs exhibit multiplicity of their genes (LysRS, SerRS, PheRS). Therefore, methanogens represent an exciting group of organisms regarding aminoacyl-tRNA synthesis, attesting to high degree of evolutionary diversity.
- Published
- 2002
19. Bioinformatic Analysis Reveals Archaeal tRNATyr and tRNATrp Identities in Bacteria
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Takahito Mukai, Noah M. Reynolds, Ana Crnković, and Dieter Söll
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tRNA ,aaRS ,genetic code ,evolution ,lateral gene transfer ,Science - Abstract
The tRNA identity elements for some amino acids are distinct between the bacterial and archaeal domains. Searching in recent genomic and metagenomic sequence data, we found some candidate phyla radiation (CPR) bacteria with archaeal tRNA identity for Tyr-tRNA and Trp-tRNA synthesis. These bacteria possess genes for tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS) predicted to be derived from DPANN superphylum archaea, while the cognate tRNATyr and tRNATrp genes reveal bacterial or archaeal origins. We identified a trace of domain fusion and swapping in the archaeal-type TyrRS gene of a bacterial lineage, suggesting that CPR bacteria may have used this mechanism to create diverse proteins. Archaeal-type TrpRS of bacteria and a few TrpRS species of DPANN archaea represent a new phylogenetic clade (named TrpRS-A). The TrpRS-A open reading frames (ORFs) are always associated with another ORF (named ORF1) encoding an unknown protein without global sequence identity to any known protein. However, our protein structure prediction identified a putative HIGH-motif and KMSKS-motif as well as many α-helices that are characteristic of class I aminoacyl-tRNA synthetase (aaRS) homologs. These results provide another example of the diversity of molecular components that implement the genetic code and provide a clue to the early evolution of life and the genetic code.
- Published
- 2017
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20. Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.
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Nikita E Chavarria, Sungmin Hwang, Shiyun Cao, Xian Fu, Mary Holman, Dina Elbanna, Suzanne Rodriguez, Deanna Arrington, Markus Englert, Sivakumar Uthandi, Dieter Söll, and Julie A Maupin-Furlow
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Medicine ,Science - Abstract
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.
- Published
- 2014
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21. The COMBREX project: design, methodology, and initial results.
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Brian P Anton, Yi-Chien Chang, Peter Brown, Han-Pil Choi, Lina L Faller, Jyotsna Guleria, Zhenjun Hu, Niels Klitgord, Ami Levy-Moonshine, Almaz Maksad, Varun Mazumdar, Mark McGettrick, Lais Osmani, Revonda Pokrzywa, John Rachlin, Rajeswari Swaminathan, Benjamin Allen, Genevieve Housman, Caitlin Monahan, Krista Rochussen, Kevin Tao, Ashok S Bhagwat, Steven E Brenner, Linda Columbus, Valérie de Crécy-Lagard, Donald Ferguson, Alexey Fomenkov, Giovanni Gadda, Richard D Morgan, Andrei L Osterman, Dmitry A Rodionov, Irina A Rodionova, Kenneth E Rudd, Dieter Söll, James Spain, Shuang-Yong Xu, Alex Bateman, Robert M Blumenthal, J Martin Bollinger, Woo-Suk Chang, Manuel Ferrer, Iddo Friedberg, Michael Y Galperin, Julien Gobeill, Daniel Haft, John Hunt, Peter Karp, William Klimke, Carsten Krebs, Dana Macelis, Ramana Madupu, Maria J Martin, Jeffrey H Miller, Claire O'Donovan, Bernhard Palsson, Patrick Ruch, Aaron Setterdahl, Granger Sutton, John Tate, Alexander Yakunin, Dmitri Tchigvintsev, Germán Plata, Jie Hu, Russell Greiner, David Horn, Kimmen Sjölander, Steven L Salzberg, Dennis Vitkup, Stanley Letovsky, Daniel Segrè, Charles DeLisi, Richard J Roberts, Martin Steffen, and Simon Kasif
- Subjects
Biology (General) ,QH301-705.5 - Published
- 2013
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22. Suppression of amber codons in Caulobacter crescentus by the orthogonal Escherichia coli histidyl-tRNA synthetase/tRNAHis pair.
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Jae-hyeong Ko, Paula Montero Llopis, Jennifer Heinritz, Christine Jacobs-Wagner, and Dieter Söll
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Medicine ,Science - Abstract
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 tRNA(His) have distinctive identity elements, we constructed E. coli tRNA(His) 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/tRNA(His) CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA(His) CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA(His) 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.
- Published
- 2013
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23. Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression
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Han-Kai Jiang, Nicole L. Ambrose, Christina Z. Chung, Yane-Shih Wang, Dieter Söll, and Jeffery M. Tharp
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Multidisciplinary - Abstract
Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.
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- 2023
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24. An Aedes aegypti seryl-tRNA synthetase paralog controls bacteroidetes growth in the midgut
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Gilbert de O. Silveira, Octávio A. C. Talyuli, Ana Beatriz Walter-Nuno, Ana Crnković, Ana C. P. Gandara, Alessandro Gaviraghi, Vanessa Bottino-Rojas, Dieter Söll, and Carla Polycarpo
- Abstract
Insect gut microbiota plays important roles in host physiology, such as nutrition, digestion, development, fertility, and immunity. We have found that in the intestine of Aedes aegypti, SLIMP (seryl-tRNA synthetase like insect mitochondrial protein) knockdown followed by a blood meal promotes dysbiosis, characterized by the overgrowth of a specific bacterial phylum, Bacteroidetes. In turn, the latter decreased both infection rates and Zika virus prevalence in the mosquitoes. Previous work in Drosophila melanogaster showed that SLIMP is involved in protein synthesis and mitochondrial respiration in a network directly coupled to mtDNA levels. There are no other reports on this enzyme and its function in other insect species. Our work expands the knowledge of the role of these SerRS paralogs. We show that A. aegypti SLIMP (AaeSLIMP) clusters with SLIMPs of the Nematocera sub-order, which have lost both the tRNA binding domain and active site residues, rendering them unable to activate amino acids and aminoacylate tRNAs. Knockdown of AaeSLIMP did not significantly influence the mosquitoes’ survival, oviposition, or eclosion. It also neither affected midgut cell respiration nor mitochondrial ROS production. However, it caused dysbiosis, which led to the activation of Dual oxidase and resulted in increased midgut ROS levels. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well.Author SummaryAminoacyl-tRNA synthetases (aaRS) are a family of ubiquitous enzymes responsible for the attachment of specific amino acids to their cognate tRNAs. During evolution some aaRS acquired new domains and/or suffered gene duplications, resulting in the improvement and expansion of their functions some of them being specific to a group of organisms. A paralog of seryl-tRNA synthetase restricted to the class Insecta (SLIMP) is found in Arthropoda. Our goal was to explore the role of SLIMP in the female mosquito Aedes aegypti using RNA interference. We showed that A. aegypti SLIMP (AaeSLIMP) gene expression is up-regulated upon blood feeding through a heme-dependent signaling. Although AaeSLIMP knockdown neither impacted the mosquito survival nor oviposition, it provoked ROS levels augmentation in the midgut via Dual Oxidase activity in order to control the increase in the intestinal native microbiota, specifically bacteria of the Bacteroidetes phylum. Although dysbiosis can result from mitochondrial impairment, this is the first time that the absence of a mitochondrial enzyme is linked to intestinal microbiota without any visible effects in mitochondrial respiration and mitochondrial ROS production. Furthermore, Zika Virus infection of AaeSLIMP silenced mosquitoes is decreased when comparing to control, meaning that Bacteroidetes overgrowth may be protecting the female mosquito. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well.
- Published
- 2022
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25. Uncovering translation roadblocks during the development of a synthetic tRNA
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Arjun Prabhakar, Natalie Krahn, Jingji Zhang, Oscar Vargas-Rodriguez, Miri Krupkin, Ziao Fu, Francisco J Acosta-Reyes, Xueliang Ge, Junhong Choi, Ana Crnković, Måns Ehrenberg, Elisabetta Viani Puglisi, Dieter Söll, and Joseph Puglisi
- Subjects
Amino Acyl-tRNA Synthetases ,RNA, Transfer ,Nucleotides ,Protein Biosynthesis ,Biochemistry and Molecular Biology ,Genetics ,Amino Acids ,Ribosomes ,Biokemi och molekylärbiologi ,Selenocysteine - Abstract
Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.
- Published
- 2022
26. Mistranslation of the genetic code by a new family of bacterial transfer RNAs
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Dominik B. Schuntermann, Jonathan T. Fischer, Jonmatthew Bile, Sarah A. Gaier, Brett A. Shelley, Aya Awawdeh, Martina Jahn, Kyle S. Hoffman, Eric Westhof, Dieter Söll, Christopher R. Clarke, and Oscar Vargas-Rodriguez
- Subjects
Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
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27. Intein-based design expands diversity of selenocysteine reporters
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Natalie Krahn, Christina Z. Chung, Dieter Söll, and Ana Crnković
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RNK ,Computational biology ,Article ,Inteins ,chemistry.chemical_compound ,Structural Biology ,Protein splicing ,Genes, Reporter ,Escherichia coli ,udc:577 ,Cysteine ,Selenoproteins ,Molecular Biology ,chemistry.chemical_classification ,biokemija ,Selenocysteine ,Directed evolution ,Recombinant Proteins ,Elongation factor ,chemistry ,genetika ,Transfer RNA ,RNA splicing ,Codon, Terminator ,Mutagenesis, Site-Directed ,proteini ,Selenoprotein ,Intein - Abstract
The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.
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- 2022
28. The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism
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Haolin Zhang, Xuemei Gong, Qianqian Zhao, Takahito Mukai, Oscar Vargas-Rodriguez, Huiming Zhang, Yuxing Zhang, Paul Wassel, Kazuaki Amikura, Julie Maupin-Furlow, Yan Ren, Xun Xu, Yuri I Wolf, Kira S Makarova, Eugene V Koonin, Yue Shen, Dieter Söll, and Xian Fu
- Subjects
Amino Acyl-tRNA Synthetases ,RNA, Transfer ,Genetic Code ,Lysine ,Genetics ,Euryarchaeota ,Amino Acids - Abstract
Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.
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- 2022
29. Indirect routes to aminoacyl-tRNA : ǂthe ǂdiversity of prokaryotic cysteine encoding systems
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Takahito Mukai, Kazuaki Amikura, Xian Fu, Dieter Söll, and Ana Crnković
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biokemija ,tRNK ,selenocysteine ,aminoacyl-tRNA synthetases ,O-phosphoseryl-tRNA synthetase ,bioinformatics ,QH426-470 ,metagenome ,aminokisline ,genetic code ,Genetics ,udc:577 ,Molecular Medicine ,proteini ,beljakovine ,tRNA ,cysteine ,Genetics (clinical) ,Original Research - Abstract
Universally present aminoacyl-tRNA synthetases (aaRSs) stringently recognize their cognate tRNAs and acylate them with one of the proteinogenic amino acids. However, some organisms possess aaRSs that deviate from the accurate translation of the genetic code and exhibit relaxed specificity toward their tRNA and/or amino acid substrates. Typically, these aaRSs are part of an indirect pathway in which multiple enzymes participate in the formation of the correct aminoacyl-tRNA product. The indirect cysteine (Cys)-tRNA pathway, originally thought to be restricted to methanogenic archaea, uses the unique O-phosphoseryl-tRNA synthetase (SepRS), which acylates the non-proteinogenic amino acid O-phosphoserine (Sep) onto tRNACys. Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNACys to SepCysS for conversion of the tRNA-bound Sep to Cys. Here, we report a comprehensive bioinformatic analysis of the diversity of indirect Cys encoding systems. These systems are present in more diverse groups of bacteria and archaea than previously known. Given the occurrence and distribution of some genes consistently flanking SepRS, it is likely that this gene was part of an ancient operon that suffered a gradual loss of its original components. Newly identified bacterial SepRS sequences strengthen the suggestion that this lineage of enzymes may not rely on the m1G37 identity determinant in tRNA. Some bacterial SepRSs possess an N-terminal fusion resembling a threonyl-tRNA synthetase editing domain, which interestingly is frequently observed in the vicinity of archaeal SepCysS genes. We also found several highly degenerate SepRS genes that likely have altered amino acid specificity. Cross-analysis of selenocysteine (Sec)-utilizing traits confirmed the co-occurrence of SepCysE and the Sec-utilizing machinery in archaea, but also identified an unusual O-phosphoseryl-tRNASec kinase fusion with an archaeal Sec elongation factor in some lineages, where it may serve in place of SepCysE to prevent crosstalk between the two minor aminoacylation systems. These results shed new light on the variations in SepRS and SepCysS enzymes that may reflect adaptation to lifestyle and habitat, and provide new information on the evolution of the genetic code.
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- 2022
30. Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo
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Jeffery M. Tharp, Omer Ad, Kazuaki Amikura, Fred R. Ward, Emma M. Garcia, Jamie H. D. Cate, Alanna Schepartz, and Dieter Söll
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General Medicine - Published
- 2020
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31. Author response: Measuring the tolerance of the genetic code to altered codon size
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Erika Alden DeBenedictis, Dieter Söll, and Kevin M Esvelt
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- 2022
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32. Using selenocysteine-specific reporters to screen for efficient tRNA
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Christina Z, Chung, Dieter, Söll, and Natalie, Krahn
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RNA, Transfer ,Protein Biosynthesis ,Escherichia coli ,RNA, Transfer, Amino Acid-Specific ,Selenoproteins ,Article ,Selenocysteine - Abstract
The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.
- Published
- 2022
33. Directed Evolution of
- Author
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Jonathan T, Fischer, Dieter, Söll, and Jeffery M, Tharp
- Abstract
Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of
- Published
- 2022
34. Khorana, Har Gobind
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Dieter Söll and Uttam RajBhandary
- Published
- 2022
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35. Using selenocysteine-specific reporters to screen for efficient tRNASec variants
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Christina Z. Chung, Dieter Söll, and Natalie Krahn
- Published
- 2022
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36. Ancestral archaea expanded the genetic code with pyrrolysine
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Li-Tao Guo, Kazuaki Amikura, Han-Kai Jiang, Takahito Mukai, Xian Fu, Yane-Shih Wang, Patrick O’Donoghue, Dieter Söll, and Jeffery M. Tharp
- Subjects
Amino Acyl-tRNA Synthetases ,RNA, Transfer ,Genetic Code ,Lysine ,Methanosarcina ,Cell Biology ,Archaea ,Molecular Biology ,Biochemistry - Abstract
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
- Published
- 2022
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37. Bacterial translation machinery for deliberate mistranslation of the genetic code
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Manyun Chen, Ahmed H. Badran, Jonathan R Krieger, Sergey Melnikov, Yousong Ding, Ana Crnković, Kyle S. Hoffman, Dieter Söll, Oscar Vargas-Rodriguez, and Eric Westhof
- Subjects
RNK ,Proline ,Sequence Homology ,RNA, Transfer, Amino Acyl ,Biology ,Substrate Specificity ,Amino Acyl-tRNA Synthetases ,Sense Codon ,chemistry.chemical_compound ,Prokaryotic translation ,udc:577 ,Escherichia coli ,Amino Acid Sequence ,Codon ,Gene ,Alanine ,Genetics ,biokemija ,Multidisciplinary ,Aminoacyl tRNA synthetase ,RNA ,Biological Sciences ,Genetic code ,Streptomyces ,streptomicete ,genetika ,chemistry ,Genetic Code ,Protein Biosynthesis ,Transfer RNA - Abstract
Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-transfer RNA (tRNA) synthetases. Using bacterial prolyl-tRNA synthetase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corresponding tRNA, tRNA(ProA), that are predominately found in plant pathogens from Streptomyces species. We then show that tRNA(ProA) has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. Finally, we provide biochemical, genetic, and mass spectrometric evidence that cells which express ProRSx and tRNA(ProA) can translate GCU alanine codons as both alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the initial example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.
- Published
- 2021
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38. The Nbp35/ApbC homolog acts as a nonessential [4Fe‐4S] transfer protein in methanogenic archaea
- Author
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Feng Long, Yuchen Liu, Taiwo S. Akinyemi, David J. Vinyard, Cuiping Zhao, William B. Whitman, Kasidet Manakongtreecheep, Dieter Söll, and Zhe Lyu
- Subjects
Iron-Sulfur Proteins ,Archaeal Proteins ,Methanococcus ,Biophysics ,Iron–sulfur cluster ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosol ,Structural Biology ,Genetics ,Nucleotide ,Molecular Biology ,Phylogeny ,030304 developmental biology ,Cell Nucleus ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Binding protein ,030302 biochemistry & molecular biology ,Methanococcus maripaludis ,Cell Biology ,biology.organism_classification ,Methanogen ,Target protein ,Gene Deletion ,Archaea - Abstract
The nucleotide binding protein 35 (Nbp35)/cytosolic Fe-S cluster deficient 1 (Cfd1)/alternative pyrimidine biosynthetic protein C (ApbC) protein homologs have been identified in all three domains of life. In eukaryotes, the Nbp35/Cfd1 heterocomplex is an essential Fe-S cluster assembly scaffold required for the maturation of Fe-S proteins in the cytosol and nucleus, whereas the bacterial ApbC is an Fe-S cluster transfer protein only involved in the maturation of a specific target protein. Here, we show that the Nbp35/ApbC homolog MMP0704 purified from its native archaeal host Methanococcus maripaludis contains a [4Fe-4S] cluster that can be transferred to a [4Fe-4S] apoprotein. Deletion of mmp0704 from M. maripaludis does not cause growth deficiency under our tested conditions. Our data indicate that Nbp35/ApbC is a nonessential [4Fe-4S] cluster transfer protein in methanogenic archaea.
- Published
- 2019
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39. Translation of Diverse Aramid- and 1,3-Dicarbonyl-peptides by Wild Type Ribosomes in Vitro
- Author
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Andrew G. Cairns, Dieter Söll, Alanna Schepartz, Scott J. Miller, Kyle S. Hoffman, Omer Ad, and Aaron L. Featherston
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biology ,010405 organic chemistry ,Chemistry ,General Chemical Engineering ,Wild type ,Ribozyme ,Translation (biology) ,General Chemistry ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Ribosome ,In vitro ,0104 chemical sciences ,Aramid ,chemistry.chemical_compound ,Biochemistry ,Biosynthesis ,Chemical Sciences ,biology.protein ,medicine ,QD1-999 ,Escherichia coli - Abstract
Here, we report that wild type Escherichia coli ribosomes accept and elongate precharged initiator tRNAs acylated with multiple benzoic acids, including aramid precursors, as well as malonyl (1,3-dicarbonyl) substrates to generate a diverse set of aramid-peptide and polyketide-peptide hybrid molecules. This work expands the scope of ribozyme- and ribosome-catalyzed chemical transformations, provides a starting point for in vivo translation engineering efforts, and offers an alternative strategy for the biosynthesis of polyketide-peptide natural products.
- Published
- 2019
- Full Text
- View/download PDF
40. Measuring the tolerance of the genetic code to altered codon size
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Erika A. DeBenedictis, Dieter Söll, and Kevin M. Esvelt
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chemistry.chemical_compound ,chemistry ,Aminoacyl tRNA synthetase ,Transfer RNA ,Translation (biology) ,Computational biology ,Alphabet ,Biology ,Protein translation ,Genetic code ,Expanded genetic code ,Selection (genetic algorithm) - Abstract
SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.
- Published
- 2021
- Full Text
- View/download PDF
41. Selective cysteine-to-selenocysteine changes in a [NiFe]-hydrogenase confirm a special position for catalysis and oxygen tolerance
- Author
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Harrison Lee, Fraser A. Armstrong, Natalie Krahn, Dieter Söll, Rhiannon M. Evans, and Bonnie J. Murphy
- Subjects
0301 basic medicine ,oxygen tolerance ,Hydrogenase ,Stereochemistry ,selenocysteine ,chemistry.chemical_element ,Overpotential ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Chalcogen ,Cysteine ,chemistry.chemical_classification ,Multidisciplinary ,Selenocysteine ,Escherichia coli Proteins ,Biological Sciences ,hydrogen activation ,0104 chemical sciences ,Oxygen ,030104 developmental biology ,Enzyme ,chemistry ,Amino Acid Substitution ,Biocatalysis ,Selenium - Abstract
Significance Substitution of cysteine by selenocysteine is held responsible for the increased performance of many enzymes: The higher activity of [NiFeSe]-hydrogenases compared with their [NiFe] counterparts is often attributed to the Sec replacement of one active-site cysteine ligand. Replacing each of the four active-site cysteine residues in an O2-tolerant [NiFe]-hydrogenase by selenocysteine shows that this substitution alone does not overcome the inability to evolve H2 that is a characteristic of the group 1d hydrogenases. A nonbridging cysteine lying on the direct path between the Ni and an adjacent proton-relaying glutamic acid emerges as being very special: Its substitution by selenocysteine confers extreme tolerance to O2 but disrupts the proton transfer pathway, providing an example of where sulfur is superior to selenium., In [NiFe]-hydrogenases, the active-site Ni is coordinated by four cysteine-S ligands (Cys; C), two of which are bridging to the Fe(CO)(CN)2 fragment. Substitution of a single Cys residue by selenocysteine (Sec; U) occurs occasionally in nature. Using a recent method for site-specific Sec incorporation into proteins, each of the four Ni-coordinating cysteine residues in the oxygen-tolerant Escherichia coli [NiFe]-hydrogenase-1 (Hyd-1) has been replaced by U to identify its importance for enzyme function. Steady-state solution activity of each Sec-substituted enzyme (on a per-milligram basis) is lowered, although this may reflect the unquantified presence of recalcitrant inactive/immature/misfolded forms. Protein film electrochemistry, however, reveals detailed kinetic data that are independent of absolute activities. Like native Hyd-1, the variants have low apparent KMH2 values, do not produce H2 at pH 6, and display the same onset overpotential for H2 oxidation. Mechanistically important differences were identified for the C576U variant bearing the equivalent replacement found in native [NiFeSe]-hydrogenases, its extreme O2 tolerance (apparent KMH2 and Vmax [solution] values relative to native Hyd-1 of 0.13 and 0.04, respectively) implying the importance of a selenium atom in the position cis to the site where exogenous ligands (H−, H2, O2) bind. Observation of the same unusual electrocatalytic signature seen earlier for the proton transfer-defective E28Q variant highlights the direct role of the chalcogen atom (S/Se) at position 576 close to E28, with the caveat that Se is less effective than S in facilitating proton transfer away from the Ni during H2 oxidation by this enzyme.
- Published
- 2021
42. Introducing Selenocysteine into Recombinant Proteins in Escherichia coli
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Natalie Krahn, Christina Z. Chung, Dieter Söll, and Corwin Miller
- Subjects
chemistry.chemical_classification ,General Immunology and Microbiology ,Selenocysteine ,integumentary system ,General Neuroscience ,Health Informatics ,Translation (biology) ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Stop codon ,Article ,Amino acid ,Medical Laboratory Technology ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Transfer RNA ,medicine ,Selenoprotein ,General Pharmacology, Toxicology and Pharmaceutics ,Escherichia coli ,Cysteine - Abstract
Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.
- Published
- 2021
43. Initiating protein synthesis with noncanonical monomers in vitro and in vivo
- Author
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Dieter Söll, Joshua A. Walker, Alanna Schepartz, and Jeffery M. Tharp
- Subjects
Biochemistry & Molecular Biology ,Carboxylic acid ,Genetic code expansion ,Article ,Amino Acyl-tRNA Synthetases ,chemistry.chemical_compound ,RNA, Transfer ,Ribosomal protein ,Translation initiation ,Escherichia coli ,Protein biosynthesis ,Peptide bond ,Amino Acids ,Synthetic biology ,Genetically encoded materials ,chemistry.chemical_classification ,Methionine ,Proteins ,Translation (biology) ,Chemical biology ,Amino acid ,Transfer ,Non-canonical amino acids ,chemistry ,Biochemistry ,Transfer RNA ,RNA ,Biochemistry and Cell Biology ,Peptides - Abstract
With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNAfMet. By mis-acylating tRNAfMet, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo. Furthermore, because the α-amine of the initiating amino acid is not required for peptide bond formation, translation can be initiated with a variety of structurally disparate carboxylic acids that bear little resemblance to traditional α-amino acids. Herein, we provide a detailed protocol to initiate in vitro protein synthesis with substituted benzoic acid and 1,3-dicarbonyl compounds. These moieties are introduced at the N-terminus of peptides by mis-acylated tRNAfMet, prepared by flexizyme-catalyzed tRNA acylation. In addition, we describe a protocol to initiate in vivo protein synthesis with aromatic noncanonical amino acids (ncAAs). This method relies on an engineered chimeric initiator tRNA that is acylated with ncAAs by an orthogonal aminoacyl-tRNA synthetase. Together, these systems are useful platforms for producing N-terminally modified proteins and for engineering the protein synthesis machinery of Escherichia coli to accept additional nonproteinogenic carboxylic acid monomers.
- Published
- 2021
- Full Text
- View/download PDF
44. Thirty Years of Collaborations with Tom
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Dieter Söll
- Published
- 2020
- Full Text
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45. Structure of E. coli Glutaminyl-tRNA Synthetase Complexed with tRNAGln and ATP at 2.8 Å Resolution
- Author
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MARK A. ROULD, JOHN J. PERONA, DIETER SÖLL, and THOMAS A. STEITZ
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- 2020
- Full Text
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46. Multiplex suppression of four quadruplet codons via tRNA directed evolution
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Ahmed H. Badran, Christina Z. Chung, Gavriela D. Carver, Erika A. DeBenedictis, and Dieter Söll
- Subjects
Base pair ,Science ,General Physics and Astronomy ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Amino Acyl-tRNA Synthetases ,Synthetic biology ,RNA, Transfer ,Anticodon ,Escherichia coli ,Multiplex ,Amino Acids ,Cloning, Molecular ,Codon ,chemistry.chemical_classification ,Multidisciplinary ,Escherichia coli Proteins ,fungi ,food and beverages ,Translation (biology) ,General Chemistry ,Genetic code ,Directed evolution ,tRNAs ,Amino acid ,RNA, Bacterial ,chemistry ,Protein Biosynthesis ,Transfer RNA ,Directed Molecular Evolution - Abstract
Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon–anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system., Genetic code expansion strategies are limited to specific codons that can be reassigned to new amino acids. Here the authors show that quadruplet-decoding tRNAs (qtRNAs) can be rapidly discovered and evolved to decode new quadruplet codons, enabling four independent decoding events in a single protein in living cells.
- Published
- 2020
47. Exploiting evolutionary trade-offs for posttreatment management of drug-resistant populations
- Author
-
David L. Stevens, Jeffery Sabina, Jin-Tao Zhang, Kevin Lee, Dieter Söll, Xian Fu, Hui Si Kwok, Yue Shen, Sergey Melnikov, and Harry Lee
- Subjects
Models, Molecular ,Protein Conformation ,Drug Resistance ,Computational biology ,Drug resistance ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,Structure-Activity Relationship ,Antibiotic resistance ,Drug Resistance, Bacterial ,medicine ,Escherichia coli ,Experimental evolution ,Multidisciplinary ,Tavaborole ,Escherichia coli Proteins ,Trade offs ,Turbidostat ,Genetic Variation ,Biological Sciences ,Biological Evolution ,Anti-Bacterial Agents ,chemistry ,Norvaline - Abstract
Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant Escherichia coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis. We then show that tavaborole-sensitive cells quickly outcompete tavaborole-resistant cells in the presence of norvaline due to the amplified cost of the molecular defect of tavaborole resistance. This finding illustrates that understanding molecular mechanisms of drug resistance allows us to effectively amplify even small evolutionary vulnerabilities of resistant cells to potentially enhance or enable adaptive therapies by accelerating posttreatment competition between resistant and susceptible cells.
- Published
- 2020
48. Versatility of synthetic tRNAs in genetic code expansion
- Author
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Dieter Söll, Ana Crnković, and Kyle S. Hoffman
- Subjects
0301 basic medicine ,RNK ,lcsh:QH426-470 ,selenocysteine ,Pyrrolysine ,non-canonical amino acids ,Computational biology ,Review ,01 natural sciences ,Ribosome ,aminokisline ,03 medical and health sciences ,chemistry.chemical_compound ,Synthetic biology ,Genetics ,udc:577 ,Genetics (clinical) ,chemistry.chemical_classification ,biokemija ,Selenocysteine ,010405 organic chemistry ,Translation (biology) ,Genetic code ,transfer RNA ,0104 chemical sciences ,Amino acid ,lcsh:Genetics ,030104 developmental biology ,genetic code expansion ,chemistry ,genetika ,Transfer RNA ,synthetic biology - Abstract
Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants.
- Published
- 2020
49. Effects of heterologous tRNA modifications on the production of proteins containing noncanonical amino acids
- Author
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Ana Crnković, Oscar Vargas-Rodriguez, Dieter Söll, and Anna Merkuryev
- Subjects
0301 basic medicine ,noncanonical amino acids ,genetic code expansion ,protein translation ,tRNA ,aminoacyl-tRNA synthetases ,posttranscriptional modifications ,phosphoserine ,TRNA modification ,Bioengineering ,medicine.disease_cause ,lcsh:Technology ,Article ,Green fluorescent protein ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,medicine ,udc:577 ,Escherichia coli ,lcsh:QH301-705.5 ,chemistry.chemical_classification ,biokemija ,Aminoacyl tRNA synthetase ,Cysteine desulfurase ,lcsh:T ,Amino acid ,030104 developmental biology ,chemistry ,Biochemistry ,lcsh:Biology (General) ,Transfer RNA - Abstract
Synthesis of proteins with noncanonical amino acids (ncAAs) enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs) and tRNAs (o-tRNAs). Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep). The incorporation of Sep into a green fluorescent protein (GFP) in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS) increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.
- Published
- 2020
50. Exploiting evolutionary trade-offs to combat antibiotic resistance
- Author
-
Jeffery Sabina, Jin-Tao Zhang, David L. Stephens, Yue Shen, Dieter Söll, Xian Fu, Hui Si Kwok, Harry Lee, Sergey Melnikov, and Kevin Lee
- Subjects
Drug ,0303 health sciences ,Experimental evolution ,Tavaborole ,030306 microbiology ,media_common.quotation_subject ,Trade offs ,Turbidostat ,Computational biology ,Biology ,Therapeutic resistance ,03 medical and health sciences ,chemistry.chemical_compound ,Antibiotic resistance ,chemistry ,Norvaline ,030304 developmental biology ,media_common - Abstract
Antibiotic resistance frequently evolves through fitness trade-offs in which the genetic alterations that confer resistance to a drug can also cause growth defects in resistant cells. Here, through experimental evolution in a microfluidics-based turbidostat, we demonstrate that antibiotic-resistant cells can be efficiently inhibited by amplifying the fitness costs associated with drug-resistance evolution. Using tavaborole-resistant E. coli as a model, we show that genetic mutations in leucyl-tRNA synthetase (that underlie tavaborole resistance) make resistant cells intolerant to norvaline, a chemical analog of leucine that is mistakenly used by tavaborole-resistant cells for protein synthesis. We then show that tavaborole-sensitive cells quickly outcompete tavaborole-resistant cells in the presence of norvaline due to the amplified cost of the molecular defect of tavaborole resistance. This finding illustrates a potentially generalizable approach for combating therapeutic resistance, prolonging the effectiveness of drugs and enabling the use of drugs that are no longer effective due to the rapid evolution of resistance.
- Published
- 2020
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