RESUMO
Protein biosynthesis is a central process in all living cells that is catalyzed by a complex molecular machineâthe ribosome. This process is termed translation because the language of nucleotides in mRNAs is translated into the language of amino acids in proteins. Transfer RNA (tRNA) molecules charged with amino acids serve as adaptors and recognize codons of mRNA in the decoding center while simultaneously the individual amino acids are assembled into a peptide chain in the peptidyl transferase center (PTC). As the nascent peptide emerges from the ribosome, it is threaded through a long tunnel referred to as a nascent peptide exit tunnel (NPET). The PTC and NPET are the sites targeted by many antibiotics and are thus of tremendous importance from a biomedical perspective and for drug development in the pharmaceutical industry.Researchers have achieved much progress in characterizing ribosomal translation at the molecular level; an impressive number of high-resolution structures of different functional and inhibited states of the ribosome are now available. These structures have significantly contributed to our understanding of how the ribosome interacts with its key substrates, namely, mRNA, tRNAs, and translation factors. In contrast, much less is known about the mechanisms of how small molecules, especially antibiotics, affect ribosomal protein synthesis. This mainly concerns the structural basis of small molecule-NPET interference with cotranslational protein folding and the regulation of protein synthesis. Growing biochemical evidence suggests that NPET plays an active role in the regulation of protein synthesis.Much-needed progress in this field is hampered by the fact that during the preparation of ribosome complexes for structural studies (i.e., X-ray crystallography, cryoelectron microscopy, and NMR spectroscopy) the aminoacyl- or peptidyl-tRNAs are unstable and become hydrolyzed. A solution to this problem is the application of hydrolysis-resistant mimics of aminoacyl- or peptidyl-tRNAs.In this Account, we present an overview of synthetic methods for the generation of peptidyl-tRNA analogs. Modular approaches have been developed that combine (i) RNA and peptide solid-phase synthesis on 3'-aminoacylamino-adenosine resins, (ii) native chemical ligations and Staudinger ligations, (iii) tailoring of tRNAs by the selective cleavage of natural native tRNAs with DNAzymes followed by reassembly with enzymatic ligation to synthetic peptidyl-RNA fragments, and (iv) enzymatic tailing and cysteine charging of the tRNA to obtain modified CCA termini of a tRNA that are chemically ligated to the peptide moiety of interest. With this arsenal of tools, in principle, any desired sequence of a stably linked peptidyl-tRNA mimic is accessible. To underline the significance of the synthetic conjugates, we briefly point to the most critical applications that have shed new light on the molecular mechanisms underlying the context-specific activity of ribosome-targeting antibiotics, ribosome-dependent incorporation of multiple consecutive proline residues, the incorporation of d-amino acids, and tRNA mischarging.Furthermore, we discuss new types of stably charged tRNA analogs, relying on triazole- and squarate (instead of amide)-linked conjugates. Those have pushed forward our mechanistic understanding of nonribosomal peptide synthesis, where aminoacyl-tRNA-dependent enzymes are critically involved in various cellular processes in primary and secondary metabolism and in bacterial cell wall synthesis.
Assuntos
RNA de Transferência , RNA , Microscopia Crioeletrônica , Aminoácidos , Biossíntese de Proteínas , Peptídeos/química , Antibacterianos/farmacologia , RNA Mensageiro , BiologiaRESUMO
Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic Nα-formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated Nα-formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained Nα-formylmethionyl conjugateâand hence the chemoselectivity of the reactionâwere verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant Nα-formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.
Assuntos
Aminoacil-RNA de Transferência , RNA , Aminoacil-RNA de Transferência/metabolismo , RNA/metabolismo , Peptídeos/química , Ribossomos/metabolismoRESUMO
Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD+), defining a novel class of NAD+ riboswitches (NAD+-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD+-II riboswitch in complex with NAD+. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD+, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD+ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD+-II motif for biotechnological applications.
Assuntos
Riboswitch , NAD/metabolismo , Filogenia , Ligantes , RNA/genéticaRESUMO
Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.
Assuntos
Neoplasias da Mama , Leucemia , Pró-Fármacos , Humanos , Feminino , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Tioguanina/farmacologia , Tioguanina/metabolismo , Nucleosídeos de PurinaRESUMO
Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.
Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMO
Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.
Assuntos
Estabilidade de RNA , RNA/química , Tubercidina/química , Pareamento de Bases , Cristalografia por Raios X , Mutagênese , Purinas/química , RNA/genética , TermodinâmicaRESUMO
This review aims at juxtaposing common versus distinct structural and functional strategies that are applied by aptamers, riboswitches, and ribozymes/DNAzymes. Focusing on recently discovered systems, we begin our analysis with small-molecule binding aptamers, with emphasis on in vitro-selected fluorogenic RNA aptamers and their different modes of ligand binding and fluorescence activation. Fundamental insights are much needed to advance RNA imaging probes for detection of exo- and endogenous RNA and for RNA process tracking. Secondly, we discuss the latest gene expression-regulating mRNA riboswitches that respond to the alarmone ppGpp, to PRPP, to NAD+, to adenosine and cytidine diphosphates, and to precursors of thiamine biosynthesis (HMP-PP), and we outline new subclasses of SAM and tetrahydrofolate-binding RNA regulators. Many riboswitches bind protein enzyme cofactors that, in principle, can catalyse a chemical reaction. For RNA, however, only one system (glmS ribozyme) has been identified in Nature thus far that utilizes a small molecule - glucosamine-6-phosphate - to participate directly in reaction catalysis (phosphodiester cleavage). We wonder why that is the case and what is to be done to reveal such likely existing cellular activities that could be more diverse than currently imagined. Thirdly, this brings us to the four latest small nucleolytic ribozymes termed twister, twister-sister, pistol, and hatchet as well as to in vitro selected DNA and RNA enzymes that promote new chemistry, mainly by exploiting their ability for RNA labelling and nucleoside modification recognition. Enormous progress in understanding the strategies of nucleic acids catalysts has been made by providing thorough structural fundaments (e.g. first structure of a DNAzyme, structures of ribozyme transition state mimics) in combination with functional assays and atomic mutagenesis.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA Catalítico/metabolismo , Ácidos Nucleicos/metabolismo , RNA Catalítico/metabolismo , Riboswitch , Aptâmeros de Nucleotídeos/química , Domínio Catalítico , DNA Catalítico/química , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , RNA Catalítico/químicaRESUMO
Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.
Assuntos
DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa , Adenosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Aprendizado de Máquina , Metilação , Oligorribonucleotídeos/química , TranscriptomaRESUMO
Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.
Assuntos
Guanosina/análogos & derivados , Análise de Sequência de RNA/métodos , Tionucleosídeos/química , Sequência de Bases , Guanosina/química , Hidrazinas/química , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coloração e RotulagemRESUMO
New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA-ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with 19F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2'-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2'-SCF3 modification being the most prominent one. A major drawback of the 2'-SCF3 group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2'-OCF3 modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2'-OCF3 labeled RNA and to investigate the impact of this modification. We present the syntheses of 2'-OCF3 adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2'-OCF3 group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3'-endo conformation of the 2'-OCF3 ribose as reflected in the 3 J (H1'-H2') coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in 19F-NMR analysis of RNA structure equilibria and of RNA-small molecule interactions. The study is complemented by a crystal structure at 0.9 Å resolution of a 27 nt hairpin RNA containing a single 2'-OCF3 group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.
RESUMO
Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. Here we report the first three-dimensional structure of the recently discovered S-adenosyl-L-methionine responsive SAM-VI riboswitch. SAM-VI adopts a unique fold and ligand pocket that are distinct from all other known SAM riboswitch classes. The ligand binds to the junctional region with its adenine tightly intercalated and Hoogsteen base-paired. Furthermore, we reveal the ligand discrimination mode of SAM-VI by additional X-ray structures of this riboswitch bound to S-adenosyl-L-homocysteine and a synthetic ligand mimic, in combination with isothermal titration calorimetry and fluorescence spectroscopy to explore binding thermodynamics and kinetics. The structure is further evaluated by analysis of ligand binding to SAM-VI mutants. It thus provides a thorough basis for developing synthetic SAM cofactors for applications in chemical and synthetic RNA biology.
Assuntos
Bifidobacterium/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/ultraestrutura , Riboswitch/genética , Cristalografia por Raios X , Ligantes , RNA Bacteriano/genética , S-Adenosilmetionina/metabolismoRESUMO
The trinucleotide repeat expansion disorders (TREDs) constitute of a group of >40 hereditary neurodegenerative human diseases associated with abnormal expansion of repeated sequences, such as CAG repeats. The pathogenic factor is a transcribed RNA or protein whose function in the cell is compromised. The disorders are progressive and incurable. Consequently, many ongoing studies are oriented at developing therapies. We have analyzed crystal structures of RNA containing CAG repeats in complex with synthetic cyclic mismatch-binding ligands (CMBLs). The models show well-defined interactions between the molecules in which the CMBLs mimic nucleobases as they form pseudo-canonical base pairs with adenosine residues and engage in extensive stacking interactions with neighboring nucleotides. The binding of ligands is associated with major structural changes of the CAG repeats, which is consistent with results of biochemical studies. The results constitute an early characterization of the first lead compounds in the search for therapy against TREDs. The crystallographic data indicate how the compounds could be further refined in future biomedical studies.
Assuntos
RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Adenosina/metabolismo , Sequência de Bases , Ligantes , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , RNA/química , Solventes , Temperatura , Raios UltravioletaRESUMO
During protein synthesis, ribosomes discriminate chirality of amino acids and prevent incorporation of D-amino acids into nascent proteins by slowing down the rate of peptide bond formation. Despite this phenomenon being known for nearly forty years, no structures have ever been reported that would explain the poor reactivity of D-amino acids. Here we report a 3.7Å-resolution crystal structure of a bacterial ribosome in complex with a D-aminoacyl-tRNA analog bound to the A site. Although at this resolution we could not observe individual chemical groups, we could unambiguously define the positions of the D-amino acid side chain and the amino group based on chemical restraints. The structure reveals that similarly to L-amino acids, the D-amino acid binds the ribosome by inserting its side chain into the ribosomal A-site cleft. This binding mode does not allow optimal nucleophilic attack of the peptidyl-tRNA by the reactive α-amino group of a D-amino acid. Also, our structure suggests that the D-amino acid cannot participate in hydrogen-bonding with the P-site tRNA that is required for the efficient proton transfer during peptide bond formation. Overall, our work provides the first mechanistic insight into the ancient mechanism that helps living cells ensure the stereochemistry of protein synthesis.
Assuntos
Peptídeos/química , Biossíntese de Proteínas/genética , Aminoacil-RNA de Transferência/química , Ribossomos/química , Aminoácidos/química , Aminoácidos/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Ligação de Hidrogênio , Peptídeos/genética , Aminoacil-RNA de Transferência/genética , Ribossomos/genéticaRESUMO
The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4â Å of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage.
Assuntos
Adenosina/metabolismo , Biocatálise , Magnésio/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Adenosina/química , Domínio Catalítico , Magnésio/química , Mutagênese , Conformação Proteica , RNA Catalítico/químicaRESUMO
Nucleobase methylations are ubiquitous posttranscriptional modifications of ribonucleic acids (RNA) that can substantially increase the structural diversity of RNA in a highly dynamic fashion with implications for gene expression and human disease. However, high throughput, deep sequencing does not generally provide information on posttranscriptional modifications (PTMs). A promising alternative approach for the characterization of PTMs, i.e. their identification, localization, and relative quantitation, is top-down mass spectrometry (MS). In this study, we have investigated how specific nucleobase methylations affect RNA ionization in electrospray ionization (ESI), and backbone cleavage in collisionally activated dissociation (CAD) and electron detachment dissociation (EDD). For this purpose, we have developed two new approaches for the characterization of RNA methylations in mixtures of either isomers of RNA or nonisomeric RNA forms. Fragment ions from dissociation experiments were analyzed to identify the modification type, to localize the modification sites, and to reveal the site-specific, relative extent of modification for each site.
Assuntos
RNA/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenosina/análogos & derivados , Adenosina/análise , Adenosina/química , Sequência de Bases , Citidina/análogos & derivados , Citidina/análise , Citidina/química , Íons , Metilação , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Uridina/análogos & derivados , Uridina/análise , Uridina/químicaRESUMO
ABSTRACT: We have developed an efficient route for the synthesis of 15N(7)-labeled adenosine as phosphoramidite building block for site- and atom-specific incorporation into RNA by automated solid-phase synthesis. Such labeled RNA is required for the evaluation of selected non-canonical base pair interactions in folded RNA using NMR spectroscopic methods.
RESUMO
We present an innovative O6 -tert-butyl/N2 -tert-butyloxycarbonyl protection concept for guanosineâ (G) phosphoramidites. This concept is advantageous for 2'-modified G building blocks because of very efficient synthetic access when compared with existing routes that usually employ O6 -(4-nitrophenyl)ethyl/N2 -acyl protection or that start from 2-aminoadenosine involving enzymatic transformation into guanosine later on in the synthetic path. The new phosphoramidites are fully compatible with 2'-O-tBDMS or TOM phosphoramidites in standard RNA solid-phase synthesis and deprotection, and provide excellent quality of tailored RNAs for the growing range of applications in RNA biophysics, biochemistry, and biology.
Assuntos
Guanosina/química , Compostos Organofosforados/química , RNA/síntese química , Ácidos/química , Adenosina/análogos & derivados , Adenosina/química , Sequência de Bases , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Nitrofenóis/química , Técnicas de Síntese em Fase SólidaRESUMO
Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.
Assuntos
Peptídeos/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas , Ribossomos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Modelos Moleculares , Peptídeos/química , Prolina/química , Ligação Proteica , RNA de Transferência de Prolina/metabolismo , Ribossomos/metabolismoRESUMO
Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals a previously unseen conformation of an eIF5A-ribosome complex and highlights a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A-ribosome association.
Assuntos
Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Hydrolysis-resistant RNA-peptide conjugates that contain a 3'-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3'-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3'-NH-peptide-modified RNAs, containing the universally conserved 3'-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle.