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16 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Architecture of Pseudomonas aeruginosa glutamyl-tRNA synthetase defines a subfamily of dimeric class Ib aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases (AaRSs) are an ancient family of structurally diverse enzymes that are divided into two major classes. The functionalities of most AaRSs are inextricably linked to their oligomeric states. While GluRSs were previously classified as monomers, the current investigation reveals that the form expressed in Pseudomonas aeruginosa is a rotationally pseudosymmetrical homodimer featuring intersubunit tRNA binding sites. Both subunits display a highly bent, "pipe strap" conformation, with the anticodon binding domain directed toward the active site. The tRNA binding sites are similar in shape to those of the monomeric GluRSs, but are formed through an approximately 180-degree rotation of the anticodon binding domains and dimerization via the anticodon and D-arm binding domains. As a result, each anticodon binding domain is poised to recognize the anticodon loop of a tRNA bound to the adjacent protomer. Additionally, the anticodon binding domain has an α-helical C-terminal extension containing a conserved lysine-rich consensus motif positioned near the predicted location of the acceptor arm, suggesting dual functions in tRNA recognition. The unique architecture of PaGluRS broadens the structural diversity of the GluRS family, and member synthetases of all bacterial AaRS subclasses have now been identified that exhibit oligomerization.

Fenwick, Michael K↗
NASA NTRS1998

Evolutionary anomalies among the aminoacyl-tRNA synthetases

Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.

Non-NASA Center↗
DOE OSTI2022

Diversification of aminoacyl-tRNA synthetase activities via genomic duplication

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.

noncanonical functions↗
DOE OSTI2023

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

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. Furthermore, 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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Indirect Routes to Aminoacyl-tRNA: The Diversity of Prokaryotic Cysteine Encoding Systems

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 tRNA Cys . Together with Sep-tRNA:Cys-tRNA synthase (SepCysS) and the adapter protein SepCysE, SepRS forms a transsulfursome complex responsible for shuttling Sep-tRNA Cys 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 m 1 G37 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-tRNA Sec 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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evolution of tRNA synthetases

The disclosure provides amino acid sequence variants of orthogonal aminoacyl-tRNA synthetases (AARSs) having increased activity and selectivity compared to previous AARSs, and methods of producing the same.

Liu, David R.↗
DOE OSTI2025

Different chemical scaffolds bind to L-phe site in Mycobacterium tuberculosis Phe-tRNA synthetase

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mt), is one of the deadliest infectious diseases. The rise of multidrug-resistant strains represents a major public health threat, requiring new therapeutic options. Bacterial aminoacyl-tRNA synthetases (aaRS) have been shown to be highly promising drug targets, including for TB treatment. These enzymes play an essential role in translating the DNA gene code into protein sequence by attaching specific amino acid to their cognate tRNAs. They have multiple binding sites that can be targeted for inhibitor discovery: amino acid binding pocket, ATP binding pocket, tRNA binding site and an editing domain. Recently we reported several high-resolution structures of M. tuberculosis phenylalanyl-tRNA synthetase (MtPheRS) complexed with tRNA Phe and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. Here, in this study, using Nucleic Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) we identified fragments that bind to MtPheRS and we determined crystal structures of their complexes with MtPheRS/tRNA Phe . All the binders interact with the L-Phe amino acid binding site. The analysis of interactions of the new compounds combined with adenylate analog structure provides insights for the rational design of antituberculosis drugs. The 3 ' arm of the tRNA Phe in all the structures was disordered with exception of one complex with D-735 compound. In this structure the 3' CCA end of the acceptor stem is observed in the editing domain of MtPheRS providing insights regarding the post-transfer editing activity of class II aaRS.

Gade, Priyanka [Univ. of Chicago, IL (United State↗
DOE OSTI2025

Thioesters Support Efficient Protein Biosynthesis by the Ribosome

Thioesters are critical chemical intermediates in numerous extant biochemical reactions and are invoked as key reagents during prebiotic peptide synthesis on an evolving Earth. Here we asked if a thioester could replace the native oxo-ester in acyl-tRNA substrates during protein biosynthesis by the ribosome. We prepared 3'-thio-3'-deoxyadenosine triphosphate in 10 steps from xylose and demonstrated that it is an effective substrate for the Escherichia coli CCA-adding enzyme, which appends 3'-thio-3'-deoxyadenosine to truncated tRNAs ending with 3'-CC. Using a variety of aminoacyl-tRNA synthetases, flexizymes, or a direct thioester exchange reaction, we prepared a suite of 3'-thio-tRNAs acylated with α- and non-α-amino acids. All were recognized and utilized by wild-type E. coli ribosomes during in vitro translation reactions to generate oligopeptides in yields commensurate with native oxo-ester tRNAs. These results indicate that thioester intermediates widely used in Nature can be co-opted to support the incorporation of natural α-amino acids as well as noncanonical monomers by the extant translational machinery for sequence-defined polymer synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation

Abstract Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3′ end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Uncovering translation roadblocks during the development of a synthetic tRNA

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-tRNA UTu1 , 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-tRNA UTu1 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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens

The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for anti-trypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Morphological and Genomic Features of the New Klosneuvirinae Isolate Fadolivirus IHUMI-VV54

Since the discovery of Mimivirus, viruses with large genomes encoding components of the translation machinery and other cellular processes have been described as belonging to the nucleocytoplasmic large DNA viruses. Recently, genome-resolved metagenomics led to the discovery of more than 40 viruses that have been grouped together in a proposed viral subfamily named Klosneuvirinae. Members of this group had genomes of up to 2.4Mb in size and featured an expanded array of translation system genes. Yet, despite the large diversity of the Klosneuvirinae in metagenomic data, there are currently only two isolates available. Here, we report the isolation of a novel giant virus known as Fadolivirus from an Algerian sewage site and provide morphological data throughout its replication cycle in amoeba and a detailed genomic characterization. The Fadolivirus genome, which is more than 1.5Mb in size, encodes 1,452 predicted proteins and phylogenetic analyses place this viral isolate as a near relative of the metagenome assembled Klosneuvirus and Indivirus. The genome encodes for 66 tRNAs, 23 aminoacyl-tRNA synthetases and a wide range of transcription factors, surpassing Klosneuvirus and other giant viruses. The Fadolivirus genome also encodes putative vacuolar-type proton pumps with the domains D and A, potentially constituting a virus-derived system for energy generation. The successful isolation of Fadolivirus will enable future hypothesis-driven experimental studies providing deeper insights into the biology of the Klosneuvirinae.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1991

Macromolecular recognition: Structural aspects of the origin of the genetic system

Theoretical simulation of prebiotic chemical processes is an invaluable tool for probing the phenomenon of evolution of life. Using computational and modeling techniques and guided by analogies from present day systems we, seek to understand the emergence of genetic apparatus, enzymatic catalysis and protein synthesis under prebiotic conditions. In one possible scenario, the RNA enzymatic reaction plays a key role in the emergence of the self-replicating and offers a clue to the onset of enzymatic catalysis prior to the existence of the protein biosynthetic machinery. Our ultimate goal is to propose a simple RNA segment which contains the specificity and catalytic activity of the contemporary RNA enzyme and which could emerge in a primordial chemical environment. To understand the mechanism of ribozyme catalyzed reactions, ab initio and semi-empirical (ZINDO) programs were used to investigate the reaction path of transphosphorylation. A special emphasis was placed on the possible catalytic and structural roles played by the coordinated magnesium cation. Both the inline and adjacent mechanisms of transphosphorylation have been studied. Another important aspect of this reaction is the identity of the functional groups which are essential for the acid base catalysis. The structural characteristics of the target helices, particularly a possible role of G center dot T pair, is under examination by molecular dynamics (MD) simulation technique. Modeling of the ancestral aminoacyl-tRNA synthetases (aRS) may provide important clues to the emergence of the genetic code and the protein synthetic machinery. Assuming that the catalytic function evolved before the elements of specific recognition of a particular amino acid, we are exploring the minimal structural requirements for the catalysis of tRNA aminoacylation. The molecular modeling system SYBYL was used for this study based on the high resolution crystallographic structures of the present day tyrosyl-adenylate:tyrRS and tRNA(Gln): ATP:glnRS complexes. The trinucleotide CCA of the 3'-end tRNA is placed into the active site pocket of tyrRS, based on the scheme of interaction between tRNA(Gln) and glnRS, and upon the stereochemistry of the tyrRS:tRNA:Tyr-AMP transition state. This provides a model of the non-specific recognition of a tRNA's 3'-end by an aRS, which might be similar to that of the ancestral aRS's. In the next step, modeling of the rest of the acceptor stem of tRNA (Tyr) with tyrRS is carried out.

Rein, Robert↗
NASA NTRS1999

Archaea: From Genomics to Physiology and the Origin of Life

This document represents a report on a meeting about Archaea. The meeting had an unusually diversified mix of topics all related to Archaea highlighting their differences and similarities with other kingdoms of life. Thus, a large number of scientists from others areas of biology participated in this conference. One-third of the speakers (11 of 33) represented laboratories whose main interests have not been archaea and who have not previously participated in similar symposia or workshops. Thus, this symposium provided a unique opportunity for archaeal researchers to interact in a wider forum. Because of the broad range of topics covered, the conference also introduced many of the participants to new areas of archaeal research. The discussions of genomics, molecular mechanisms of transcription, metabolic pathways and evolution were at a very high level. Talks and posters provided detailed discussions of the state of the current knowledge in RNA processing, transcriptional initiation, chromatin structure, aminoacyl-tRNA synthetases, autotrophic CO2 fixation, Upid biosynthesis and a wide range of other topics. In addition to providing overviews, major areas of scientific argument were clearly delineated, particularly in the discussions of genomics and evolution. Some of the questions raised included: how representative are individual gene trees of organismal evolution, how prevalent is horizontal evolution, how reliable are functional assignments in genomics? On these topics, the different points of view were well represented. The future of any field depends on the enthusiasm and intellectual engagement of young scientists working in the area. Therefore, the participation of 29 graduate and postdoctoral students (out of about 135 participants) was a highlight of the meeting. This was the consequence of funding contributions by NSF and NASA.

Vothknecht, Ute C.↗
DOE OSTI2022

Ancestral archaea expanded the genetic code with pyrrolysine

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 Pyl ) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNA Pyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNA Pyl and reveal the emergence of tRNA Pyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNA Pyl species with the more common G73-containing tRNA Pyl will enable future efforts to engineer PylRS systems for further genetic code expansion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism

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.

59 BASIC BIOLOGICAL SCIENCES↗