RESUMEN
The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Lisina/análogos & derivados , Ingeniería de Proteínas/métodos , Aminoacil-ARN de Transferencia/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Codón de Terminación , Escherichia coli/genética , Genes Reporteros , Células HEK293 , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Lisina/genética , Lisina/metabolismo , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , Aminoacil-ARN de Transferencia/síntesis química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.
Asunto(s)
ADN Catalítico/metabolismo , Farmacorresistencia Bacteriana , Macrólidos/farmacología , Péptidos , Estabilidad del ARN , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia de Valina/química , Antibacterianos/farmacología , Secuencia de Bases , Escherichia coli , Espectrometría de Masas , Modelos Moleculares , Conformación de Ácido Nucleico , Fenol/química , Fosforilación , ARN Bacteriano/metabolismo , ARN de Transferencia de Valina/síntesis química , ARN de Transferencia de Valina/aislamiento & purificación , ARN de Transferencia de Valina/metabolismoRESUMEN
RNA-peptide conjugates that mimic amino acid-charged tRNAs and peptidyl-tRNAs are of high importance for structural and functional investigations of ribosomal complexes. Here, we present the synthesis of glycyl-, alanyl-, and isoleucyladenosine modified solid supports that are eligible for the synthesis of stable 3'-aminoacyl- and 3'-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work significantly expands the range of accessible peptidyl-tRNA mimics for ribosomal studies.
Asunto(s)
Poliestirenos/química , Aminoacil-ARN de Transferencia/síntesis química , ARN/química , Adenosina/química , Alanina/química , Glicina/química , Hidrólisis , Isoleucina/química , Péptidos/química , Aminoacil-ARN de Transferencia/química , Ribosomas/metabolismo , Técnicas de Síntesis en Fase SólidaRESUMEN
Aminoacyl-tRNAs have important roles in a variety of biological processes. Here, we describe the synthesis of stable aminoacyl-tRNA analogs containing 1,4-substituted 1,2,3-triazole rings. The procedure involves (i) copper-catalyzed cycloadditions of 3'-or 2'-azido-adenosine and alkynes, (ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and (iii) the enzymatic ligation of the 2'- or 3'-triazole-dinucleotides with a 22-nt RNA microhelix that mimics the acceptor arm of tRNA. Each nucleoside and nucleotide intermediate was characterized by MS spectrometry and (1)H, (31)P, and (13)C NMR spectroscopy, and the tRNA-analogs were assayed for inhibition of FemXWv, an alanyl-transferase essential for the formation of the peptidoglycan network of Gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 µM) indicate that the five-membered triazole rings acted as an isosteres of esters and can be used for the design of stable aminoacyl-tRNA analogs.
Asunto(s)
Aminoacil-ARN de Transferencia/síntesis química , Antibacterianos/síntesis química , Aminoacil-ARN de Transferencia/química , Análisis Espectral , Relación Estructura-Actividad , Transferasas/antagonistas & inhibidores , Triazoles/químicaRESUMEN
Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes.
Asunto(s)
Lantano/química , Oocitos/metabolismo , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia/síntesis química , Xenopus/metabolismo , Acilación , Animales , Codón sin Sentido/genética , Ratones , Estructura Molecular , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/química , Xenopus/genéticaRESUMEN
A fragment-based approach for the synthesis of ligands of tRNA(Lys) (3), the HIV reverse-transcription primer, is described. The use of NMR spectroscopy has proved to be very useful in this approach, not only to detect low-affinity complexes between small compounds and RNA, but also to provide information on their binding mode and on the way they can be connected. This NMR-spectroscopy-guided analysis enabled us to design micromolar ligands after the optimisation and connection of millimolar fragments with an appropriate linker. The influence of the linker region on the binding affinity and selectivity outlines the importance of having a flexible assemblage strategy with a variety of linkers in such an approach.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Fragmentos de Péptidos/química , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia/síntesis química , Secuencia de Bases , Sitios de Unión , Diseño de Fármacos , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/químicaRESUMEN
Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.
Asunto(s)
Imitación Molecular , Péptidos/química , Aminoacil-ARN de Transferencia/síntesis química , ARN/química , Secuencia de Aminoácidos , Secuencia de Bases , Hidrólisis , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/químicaRESUMEN
We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3'-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.
Asunto(s)
Carbocianinas/química , Colorantes Fluorescentes/química , ARN de Transferencia/síntesis química , Uridina/química , Transferencia Resonante de Energía de Fluorescencia , Métodos , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/metabolismo , Péptidos/metabolismo , Poli U/metabolismo , Biosíntesis de Proteínas , ARN de Hongos/síntesis química , ARN de Hongos/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Aminoacil-ARN de Transferencia/síntesis química , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Metionina/síntesis química , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.
Asunto(s)
Bacterias/enzimología , Pared Celular/química , Transferasas de Grupos Nitrogenados/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Alanina/metabolismo , Pared Celular/metabolismo , Glicina/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , Aminoacil-ARN de Transferencia/síntesis química , Serina/metabolismo , Estereoisomerismo , Especificidad por Sustrato , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
Aminoacyl-tRNAs have important roles in a variety of biological processes, including protein synthesis by ribosomes, targeting of proteins for degradation by the proteasome, and bacterial cell wall synthesis. Here we describe the synthesis of stable aminoacyl-tRNA analogues containing 1,4- and 1,5-substituted 1,2,3-triazole rings. The procedure involves i) Cu- and Ru-catalysed cycloadditions of 3'-azidoadenosine and alkynes, which produced the 1,4 and 1,5 regioisomers of the triazoles, respectively, ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and iii) the enzymatic ligation of the substituted dinucleotides with a 22 nt RNA microhelix that mimics the acceptor arm of tRNA. Nucleoside and nucleotide compounds were characterized by MS spectrometry and (1)H, (31)P and (13)C NMR spectroscopy and were assayed for inhibition of FemX(Wv), an alanyltransferase essential for the formation of the peptidoglycan network of gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 microM) indicate that the five-membered triazole rings acted as bioisosters of esters and can be used for the design of stable aminoacyl-tRNA analogues.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Aminoacil-ARN de Transferencia/síntesis química , Triazoles/química , Secuencia de Bases , Ésteres , Concentración 50 Inhibidora , Modelos Biológicos , Modelos Químicos , Estructura Molecular , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/farmacología , Relación Estructura-ActividadRESUMEN
In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.
Asunto(s)
Aminoacil-ARN de Transferencia/análisis , Aminoacil-ARN de Transferencia/síntesis química , Aminoacilación de ARN de Transferencia , Electroforesis en Gel de Poliacrilamida/métodos , Aminoacil-ARN de Transferencia/aislamiento & purificación , ARN de Transferencia de Metionina/síntesis químicaRESUMEN
The detailed synthesis of (bis)aminoacyl-pdCpAs and the corresponding singly and tandemly activated tRNAs is reported. The synthetic pathway leading to these compounds has been validated for simple amino acid residues as well as for amino acids bearing more complex side chains. Protection/deprotection strategies are described. For the bisaminoacylated tRNAs, both the synthesis of tRNAs bearing the same amino acid residue at the 2' and 3' positions and tRNAs bearing two different aminoacyl moieties are reported. Further, it is shown that the tandemly activated tRNAs are able to participate in protein synthesis.
Asunto(s)
Oligorribonucleótidos/química , Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia/metabolismo , Aminoacilación de ARN de Transferencia , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNACUA. These included Ngamma-methylarginine, Ngamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing Ngamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.
Asunto(s)
Arginina/análogos & derivados , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Arginina/síntesis química , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Luciferasas de Luciérnaga/genética , Modelos Moleculares , Aminoacil-ARN de Transferencia/síntesis químicaRESUMEN
The ability to introduce non-natural amino acids into proteins opens up new vistas for the study of protein structure and function. This approach requires suppressor tRNAs that deliver the non-natural amino acid to a ribosome associated with an mRNA containing an expanded codon. The suppressor tRNAs must be absolutely protected from aminoacylation by any of the aminoacyl-tRNA synthetases in the protein synthesizing system, or a natural amino acid will be incorporated instead of the non-natural amino acid. Here, we found that some tRNAs with non-standard structures could work as efficient four-base suppressors fulfilling the above orthogonal conditions. Using these tRNAs, we successfully demonstrated incorporation of three different non-natural amino acids into a single protein.
Asunto(s)
Aminoácidos/química , Ingeniería de Proteínas/métodos , Proteínas/síntesis química , Proteínas/genética , ARN de Transferencia/metabolismo , Secuencia de Aminoácidos , Aminoacilación , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Secuencia de Bases , Codón/química , Codón/genética , Codón/metabolismo , Estructura Molecular , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/síntesis química , Supresión GenéticaRESUMEN
Here we describe a novel technology that allows users to charge nonnatural amino acids onto any tRNA. This technology is based on a resin-immobilized ribozyme system, called Flexiresin. It enables users to readily and rapidly synthesize misacylated tRNAs with a wide variety of phenylalanine analogs. Since Flexiresin is reusable and little effort is necessary for regeneration, it is economical and convenient. Moreover, it can adapt to virtually any tRNA chosen by the user, and can therefore be applied to not only a single site mutation but also multiple sites with designated nonnatural amino acids when both the amber and programmed frame-shift mutations are utilized. The original ribozyme utilized for Flexiresin was artificially generated in vitro, and thus the technology in principle could be broadened from Phe analogues to essentially any amino acid.
Asunto(s)
Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , ARN Catalítico/química , Aminoacil-ARN de Transferencia/síntesis química , Aminoacilación de ARN de Transferencia , Aminoácidos/química , ARN de Transferencia/química , Moldes Genéticos , Transcripción GenéticaRESUMEN
The elaboration of misacylated transfer RNAs by T4 RNA ligase-mediated condensation of an aminoacylated pdCpA derivative and a tRNA (transcript) missing the two 3'-terminal nucleotides requires that the aminoacyl moiety of the dinucleotide be stabilized during the ligation reaction. This can be done conveniently by the use of a simple 4-pentenoyl group attached to N(alpha) of the amino acid. The pentenoyl amide can be deblocked readily with aqueous iodine, presumably via an iodolactone intermediate. This protecting group can be used in conjunction with side chain protecting group for amino acids having side chain functionality, thus permitting the elaboration of proteins bearing side chain protecting groups that can be removed in a subsequent step (e.g., caged proteins). In addition, an aminated analogue of the pentenoyl protecting group, the unnatural amino acid allylglycine, can be employed as part of the peptide backbone to afford a protein cleavable by iodine.
Asunto(s)
Aminoacil-ARN de Transferencia/síntesis química , ARN de Transferencia/química , Aminoacilación de ARN de Transferencia , Conformación de Ácido Nucleico , ARN Ligasa (ATP)/química , Proteínas Virales/químicaRESUMEN
Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.
Asunto(s)
Anticodón/química , Codón/química , Ácidos Nucleicos de Péptidos/química , Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , Aminoacilación de ARN de Transferencia , Aminoácidos/química , ARN de Transferencia , Aminoacil-ARN de Transferencia/síntesis químicaRESUMEN
It is appealing to envision engineering translation for the genetically encoded synthesis of new classes of molecules. The complete reassignment of codons to unnatural amino acids at one or two non-adjacent sites per protein has already found wide utility (see other papers in this volume). This has been achieved by suppression at stop codons or rarely used sense codons in crude systems and in vivo. However, competing aminoacyl-tRNAs, aminoacyl-tRNA synthetases, and release factors limit efficiencies and generalization. We maximize flexibility by omitting the competing components and by reconstituting translation from His-tagged initiation and elongation factors. This approach opens up all 64 codons to amino acid reassignment and has allowed incorporation of several adjacent unnatural amino acids for the study of translation mechanism. One potential application is "peptidomimetic evolution" for ligand discovery. Toward this goal, we have demonstrated the display of polypeptides on their mRNAs in a purified translation system, termed "pure translation display."