Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.116
Filtrar
1.
J Am Chem Soc ; 146(10): 6544-6556, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38426740

RESUMO

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.


Assuntos
Lisina , Lisina/análogos & derivados , S-Adenosilmetionina , Lisina/química , Código Genético , Amidas/metabolismo
2.
Protein Sci ; 33(4): e4953, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38511490

RESUMO

Deciphering the structural effects of gene variants is essential for understanding the pathophysiological mechanisms of genetic diseases. Using a neurodevelopmental disorder called Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) as a genetic disease model, we applied structural bioinformatics and Genetic Code Expansion (GCE) strategies to assess the pathogenic impact of human NR2F1 variants and their binding with known and novel partners. While the computational analyses of the NR2F1 structure delineated the molecular basis of the impact of several variants on the isolated and complexed structures, the GCE enabled covalent and site-specific capture of transient supramolecular interactions in living cells. This revealed the variable quaternary conformations of NR2F1 variants and highlighted the disrupted interplay with dimeric partners and the newly identified co-factor, CRABP2. The disclosed consequence of the pathogenic mutations on the conformation, supramolecular interplay, and alterations in the cell cycle, viability, and sub-cellular localization of the different variants reflect the heterogeneous disease spectrum of BBSOAS and set up novel foundation for unveiling the complexity of neurodevelopmental diseases.


Assuntos
Deficiência Intelectual , Humanos , Mutação , Deficiência Intelectual/genética , Código Genético
3.
J Am Chem Soc ; 146(12): 8058-8070, 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38491946

RESUMO

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 µM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.


Assuntos
Aminoácidos , Peptídeos , Humanos , Peptídeos/química , Aminoácidos/química , Código Genético , RNA Mensageiro
4.
Chimia (Aarau) ; 78(1-2): 22-31, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38430060

RESUMO

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.


Assuntos
Aminoacil-tRNA Sintetases , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Código Genético , Engenharia de Proteínas/métodos , Aminoácidos/genética , Aminoácidos/química , RNA de Transferência/genética
5.
Methods Mol Biol ; 2760: 219-251, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468092

RESUMO

Expanding the genetic code beyond the 20 canonical amino acids enables access to a wide range of chemical functionality that is inaccessible within conventionally biosynthesized proteins. The vast majority of efforts to expand the genetic code have focused on the orthogonal translation systems required to achieve the genetically encoded addition of noncanonical amino acids (ncAAs) into proteins. There remain tremendous opportunities for identifying genetic and genomic factors that enhance ncAA incorporation. Here we describe genome-wide screening strategies to identify factors that enable more efficient addition of ncAAs to biosynthesized proteins. These unbiased screens can reveal previously unknown genes or mutations that can enhance ncAA incorporation and deepen our understanding of the translation apparatus.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas/química , Código Genético , Aminoacil-tRNA Sintetases/metabolismo
6.
Methods Mol Biol ; 2760: 209-217, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468091

RESUMO

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.


Assuntos
Aminoacil-tRNA Sintetases , Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Código Genético , Aminoácidos/genética , Aminoácidos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/metabolismo
7.
Nat Chem Biol ; 20(4): 406-407, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38467845
8.
Biosystems ; 237: 105135, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38320621

RESUMO

The existent algebraic models of the genetic code contribute to the understanding of the physio-chemical characteristics of the amino acids. However, the process of translating a gene into a phenotype is highly complex. Moreover, the intricacy of gene expression gets further multiplied due to the biases in the codon usage. This paper explores an algebraic structure called module on the set of codons as well as on that of RNA sequences. We study the potential implications of these structures on gene expression and the GC content of an RNA sequence. The base order {C,U,G,A} appears to possess greater biological significance than many of the orders previously studied. We have developed a novel algorithm to generate RNA sequences with high GC content, aiming to enhance the thermostability of biomolecules. The insights gained from this investigation may have applications in biomolecular modeling and docking, protein engineering, drug development, and related fields.


Assuntos
Código Genético , Sequência de Bases , Composição de Bases , Código Genético/genética , Códon/genética , Expressão Gênica
9.
Biosystems ; 237: 105133, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38336225

RESUMO

Life codes increase in both number and variety with biological complexity. Although our knowledge of codes is constantly expanding, the evolutionary progression of organic, neural, and cultural codes in response to selection pressure remains poorly understood. Greater clarification of the selective mechanisms is achieved by investigating how major evolutionary transitions reduce spatiotemporal and energetic constraints on transmitting heritable code to offspring. Evolution toward less constrained flows is integral to enduring flow architecture everywhere, in both engineered and natural flow systems. Beginning approximately 4 billion years ago, the most basic level for transmitting genetic material to offspring was initiated by protocell division. Evidence from ribosomes suggests that protocells transmitted comma-free or circular codes, preceding the evolution of standard genetic code. This rudimentary information flow within protocells is likely to have first emerged within the geo-energetic and geospatial constraints of hydrothermal vents. A broad-gauged hypothesis is that major evolutionary transitions overcame such constraints with tri-flow adaptations. The interconnected triple flows incorporated energy-converting, spatiotemporal, and code-based informational dynamics. Such tri-flow adaptations stacked sequence splicing code on top of protein-DNA recognition code in eukaryotes, prefiguring the transition to sexual reproduction. Sex overcame the spatiotemporal-energetic constraints of binary fission with further code stacking. Examples are tubulin code and transcription initiation code in vertebrates. In a later evolutionary transition, language reduced metabolic-spatiotemporal constraints on inheritance by stacking phonetic, phonological, and orthographic codes. In organisms that reproduce sexually, each major evolutionary transition is shown to be a tri-flow adaptation that adds new levels of code-based informational exchange. Evolving biological complexity is also shown to increase the nongenetic transmissibility of code.


Assuntos
Eucariotos , Código Genético , Animais , Código Genético/genética , Eucariotos/genética , Vertebrados/genética , Reprodução , Ribossomos , Evolução Molecular
10.
Artif Life ; 30(1): 16-27, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38358121

RESUMO

In the mid-20th century, two new scientific disciplines emerged forcefully: molecular biology and information-communication theory. At the beginning, cross-fertilization was so deep that the term genetic code was universally accepted for describing the meaning of triplets of mRNA (codons) as amino acids. However, today, such synergy has not taken advantage of the vertiginous advances in the two disciplines and presents more challenges than answers. These challenges not only are of great theoretical relevance but also represent unavoidable milestones for next-generation biology: from personalized genetic therapy and diagnosis to Artificial Life to the production of biologically active proteins. Moreover, the matter is intimately connected to a paradigm shift needed in theoretical biology, pioneered a long time ago, that requires combined contributions from disciplines well beyond the biological realm. The use of information as a conceptual metaphor needs to be turned into quantitative and predictive models that can be tested empirically and integrated in a unified view. Successfully achieving these tasks requires a wide multidisciplinary approach, including Artificial Life researchers, to address such an endeavour.


Assuntos
Biologia , Código Genético
11.
BMC Genomics ; 25(1): 184, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365628

RESUMO

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.


Assuntos
Parasitos , Trypanosoma brucei brucei , Trypanosomatina , Animais , Códon de Terminação , Eucariotos/genética , Código Genético , Parasitos/genética , Trypanosoma brucei brucei/genética , Trypanosomatina/genética
12.
Biosystems ; 237: 105159, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38373543

RESUMO

I support the hypothesis that the origin of the genetic code occurred simultaneously with the evolution of cellularity. That is to say, I favour the hypothesis that the origin of the genetic code is a very, very late event in the history of life on Earth. I corroborate this hypothesis with observations favouring the progenote's stage for the Last Universal Common Ancestor (LUCA), for the ancestor of bacteria and that of archaea. Indeed, these progenotic stages would imply that - at that time - the origin of the genetic code was still ongoing simply because this origin would fall within the very definition of progenote. Therefore, if the evolution of cellularity had truly been coeval with the origin of the genetic code - at least in its terminal part - then this would favour theories such as the coevolution theory of the origin of the genetic code because this theory would postulate that this origin must have occurred in extremely complex protocellular conditions and not concerning stereochemical or physicochemical interactions having to do with other stages of the origin of life. In this sense, the coevolution theory would be corroborated while the stereochemical and physicochemical theories would be damaged. Therefore, the origin of the genetic code would be linked to the origin of the cell and not to the origin of life as sometimes asserted. Therefore, I will discuss the late hypothesis of the origin of the genetic code in the context of the theories proposed to explain this origin and more generally of its implications for the early evolution of life.


Assuntos
Evolução Molecular , Código Genético , Código Genético/genética , Bactérias/genética , Archaea/genética
14.
ACS Chem Biol ; 19(2): 516-525, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38277773

RESUMO

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.


Assuntos
Aminoácidos , Proteínas , Animais , Xenopus laevis/genética , Xenopus laevis/metabolismo , Aminoácidos/química , Proteínas/metabolismo , Código Genético , Relação Estrutura-Atividade
15.
RNA Biol ; 21(1): 1-8, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38169326

RESUMO

Preexisting partial genetic codes can fuse to evolve towards the complete Standard Genetic Code (SGC). Such code fusion provides a path of 'least selection', readily generating precursor codes that resemble the SGC. Consequently, such least selections produce the SGC via minimal, thus rapid, change. Optimal code evolution therefore requires delayed wobble. Early wobble encoding slows code evolution, very specifically diminishing the most likely SGC precursors: near-complete, accurate codes which are the products of code fusions. In contrast: given delayed wobble, the SGC can emerge from a truncation selection/evolutionary radiation based on proficient fused coding.


Assuntos
Evolução Molecular , Código Genético , Códon , Anticódon , Modelos Genéticos
16.
Mol Cell ; 84(1): 94-106, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38181765

RESUMO

RNA molecules are modified post-transcriptionally to acquire their diverse functions. Transfer RNA (tRNA) has the widest variety and largest numbers of RNA modifications. tRNA modifications are pivotal for decoding the genetic code and stabilizing the tertiary structure of tRNA molecules. Alternation of tRNA modifications directly modulates the structure and function of tRNAs and regulates gene expression. Notably, thermophilic organisms exhibit characteristic tRNA modifications that are dynamically regulated in response to varying growth temperatures, thereby bolstering fitness in extreme environments. Here, we review the history and latest findings regarding the functions and biogenesis of several tRNA modifications that contribute to the cellular thermotolerance of thermophiles.


Assuntos
Termotolerância , Termotolerância/genética , Processamento Pós-Transcricional do RNA , Código Genético , RNA de Transferência/genética , RNA/genética
17.
Science ; 383(6680): 247, 2024 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-38236976
18.
Chem Commun (Camb) ; 60(12): 1607-1610, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38230513

RESUMO

Extensive research has focused on genetic code reprogramming using flexizymes (Fxs), ribozymes enabling diverse tRNA acylation. Here we describe a nucleoside-modification strategy for the preparation of flexizyme variants derived from 2'-OMe, 2'-F, and 2'-MOE modifications with unique and versatile activities, enabling the charging of tRNAs with a broad range of substrates. This innovative strategy holds promise for synthetic biology applications, offering a robust pathway to expand the genetic code for diverse substrate incorporation.


Assuntos
RNA Catalítico , Aminoacilação de RNA de Transferência , Nucleosídeos/metabolismo , RNA de Transferência/metabolismo , Código Genético , RNA Catalítico/metabolismo
19.
Science ; 383(6679): 146-147, 2024 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-38207031
20.
Nature ; 625(7995): 603-610, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38200312

RESUMO

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Escherichia coli , Código Genético , RNA de Transferência , Acilação , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Código Genético/genética , Hidroxiácidos/química , Hidroxiácidos/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Ribossomos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...