RESUMEN
The genetic code-the language used by cells to translate their genomes into proteins that perform many cellular functions-is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.
Asunto(s)
Código Genético , Ingeniería Metabólica/métodos , Aminoácidos , Biotecnología/métodos , Codón , Biosíntesis de ProteínasRESUMEN
We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.
Asunto(s)
ARN Bacteriano/química , ARN de Transferencia/química , Anticodón , Bacterias/genética , Toxinas Bacterianas/genética , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismoRESUMEN
In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNASec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNASec-like tRNA genes distributed across Bacteria that also encode a canonical tRNASec. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNASec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNASec-like tRNACys, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNACys as 'tRNAReC' (Recoding with Cys). We performed a comprehensive survey of tRNAReC genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNAReC had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNASec. We renamed this unusual tRNASec derived from tRNAReC as 'tRNAReU' (Recoding with Sec). Together, our results suggest that tRNAReC and tRNAReU may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Proteínas Bacterianas/genética , Cisteína/metabolismo , Escherichia coli/genética , ARN de Transferencia de Cisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Anticodón/genética , Anticodón/metabolismo , Proteínas Bacterianas/metabolismo , Codón de Terminación/química , Codón de Terminación/metabolismo , Desulfotomaculum/genética , Desulfotomaculum/metabolismo , Escherichia coli/metabolismo , Código Genético , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Peptococcaceae/genética , Peptococcaceae/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia de Cisteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Selenoproteínas/biosíntesisRESUMEN
BACKGROUND: Development of new aminoacyl-tRNA synthetase (aaRS)â¢tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNAHis recognition that prevent their cross-reactivity. Although the E. coli HisRSâ¢tRNAHis pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus. METHODS: E. coli was genetically engineered to use a C. crescentus HisRSâ¢tRNAHis pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo. RESULTS: A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRSâ¢tRNAHis pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRSâ¢tRNAHis pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUA elevated its suppression efficiency by 2-fold. CONCLUSIONS: The C. crescentus HisRSâ¢tRNAHis pair functionally complements an E. coli ΔhisS strain. The E. coli HisRSâ¢tRNAHis is orthogonal in MEOV1 cells. E. coli tRNAHisCUA is an efficient amber suppressor in MEOV1. GENERAL SIGNIFICANCE: We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Histidina-ARNt Ligasa/metabolismo , Ingeniería de Proteínas/métodos , ARN de Transferencia de Histidina/metabolismo , Clonación Molecular/métodos , Biblioteca de Genes , Ingeniería Genética/métodos , Histidina-ARNt Ligasa/genética , Mutagénesis Sitio-Dirigida , ARN de Transferencia de Histidina/genéticaRESUMEN
Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRSâ¢Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.
Asunto(s)
Alanina/análogos & derivados , Staphylococcus aureus , Tiofenos/química , Tiorredoxinas/química , Triptófano/química , Alanina/química , Alanina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Dominio Catalítico , Cinética , Modelos Moleculares , Sondas Moleculares , Ingeniería de Proteínas , Staphylococcus aureus/genética , Tiofenos/metabolismo , Aminoacilación de ARN de TransferenciaRESUMEN
Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.
Asunto(s)
Factor Tu de Elongación Peptídica/genética , Selenocisteína/metabolismo , Selenoproteínas/biosíntesis , Asparagina/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Cisteína/química , Humanos , Mutación , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Ingeniería de Proteínas , ARN de Transferencia Aminoácido-Específico/metabolismoRESUMEN
Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins inâ vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin typeâ 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.
Asunto(s)
Enzimas/química , Lisina/química , Acetilación , Reparación del ADN , Replicación del ADN , Espectrometría de Masas en TándemRESUMEN
Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4â trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E.â coli to synthesize active formate dehydrogenaseâ H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.
Asunto(s)
Bacterias/genética , Codón de Terminación/genética , Genoma Bacteriano , Selenocisteína/genética , Secuencia de Bases , Evolución Molecular , Código Genético , MetagenomaRESUMEN
The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2',3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Proteínas de Escherichia coli/química , Polinucleótido Ligasas/química , Polinucleótido Ligasas/genética , Pyrococcus horikoshii/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , GMP Cíclico/química , Guanosina Trifosfato/química , Iones , Manganeso/química , Modelos Moleculares , Conformación Molecular , Unión Proteica , Empalme del ARN , ARN de Transferencia/química , Especificidad por Sustrato , Sulfatos/químicaRESUMEN
Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3'-halves beginning with a 5'-hydroxyl, and 5'-halves ending in a 2',3'-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3'-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2',3'-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3'-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn(2+) binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3'-P RNA ligase.
Asunto(s)
Proteínas Arqueales/metabolismo , Euryarchaeota/enzimología , ARN Ligasa (ATP)/metabolismo , Precursores del ARN/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Secuencia de Bases , Sitios de Unión , Biocatálisis , Euryarchaeota/genética , Datos de Secuencia Molecular , Mutación , Fosfatos/metabolismo , Filogenia , Pyrococcus horikoshii/enzimología , Pyrococcus horikoshii/genética , ARN Ligasa (ATP)/clasificación , ARN Ligasa (ATP)/genética , Precursores del ARN/genética , Empalme del ARN , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Homología de Secuencia de Aminoácido , Zinc/metabolismoRESUMEN
Animal cells have two tRNA splicing pathways: (i) a 5'-P ligation mechanism, where the 5'-phosphate of the 3' tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3'-P ligation process, in which the 3'-phosphate of the 5' tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2'-phosphate terminus for the ligation of RNAs with 5'-phosphate and 3'-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5'-P RNA ligation pathway in an animal organism.
Asunto(s)
Cordados/metabolismo , ARN Ligasa (ATP)/metabolismo , Empalme del ARN , ARN de Transferencia/metabolismo , Animales , Secuencia de Bases , Cordados/genética , Análisis Mutacional de ADN , Genes Letales , Prueba de Complementación Genética , Filogenia , ARN Ligasa (ATP)/clasificación , ARN Ligasa (ATP)/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains--an N-terminal ligase component and a C-terminal 5'-kinase/2',3'-cyclic phosphodiesterase (CPD) component--that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure-function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2'-PO4 end for tRNA splicing in vivo.
Asunto(s)
ARN Ligasa (ATP)/química , ARN Ligasa (ATP)/metabolismo , Empalme del ARN , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/química , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dominio Catalítico , Análisis Mutacional de ADN , Prueba de Complementación Genética , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/química , ARN Ligasa (ATP)/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting.
Asunto(s)
Endorribonucleasas/metabolismo , Fosfotransferasas/metabolismo , Células Vegetales , Plantas/enzimología , ARN Ligasa (ATP)/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/enzimología , Secuencia de Bases , Núcleo Celular/enzimología , Estructuras Celulares/enzimología , Cloroplastos/enzimología , Citoplasma/enzimología , Mitocondrias/enzimología , Datos de Secuencia Molecular , Cebollas/citología , Cebollas/enzimología , Oryza/citología , Oryza/enzimología , Epidermis de la Planta/citología , Plantas/genética , Transporte de Proteínas , Precursores del ARN/genética , ARN de Planta/metabolismo , Vicia faba/citología , Vicia faba/enzimologíaRESUMEN
Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.
Asunto(s)
Filogenia , Plantas/enzimología , ARN Ligasa (ATP)/clasificación , ARN Ligasa (ATP)/metabolismo , ARN de Transferencia/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Genes de Plantas , Datos de Secuencia Molecular , ARN Ligasa (ATP)/genética , Empalme del ARN , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Triticum/enzimologíaRESUMEN
Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Azufre/metabolismo , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/metabolismo , Adenosina Trifosfato/metabolismo , Cisteína/fisiología , Haloferax volcanii/enzimología , Ligandos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Compuestos de Sulfhidrilo/metabolismo , Termodinámica , UbiquitinaciónRESUMEN
In the human nuclear genome only a few copies coding for full-length 7SL RNA genes exist. The Hs7SL-1 gene has recently been classified as type 4 of RNA polymerase III (pol III)-transcribed genes as it was demonstrated that mutations in an external transcriptional activator (ATF) binding site and in an internal CG dinucleotide at positions +15/+16 reduced 7SL RNA expression in vivo and in vitro. We have extended the elucidation of external and internal promoter elements and have discovered two novel regulatory sequences: a TATA-like element in the upstream region and internal A and B box-like motifs. This study was greatly facilitated by the identification of a second, new functional human 7SL RNA gene which we called Hs7SL-3. Remarkably, Hs7SL-3 RNA is synthesized twice as efficiently as Hs7SL-1 in HeLa nuclear extract. Comparison of the upstream regions revealed the presence of two conserved elements in the two human 7SL RNA genes, an ATF/CRE binding site at -43 to -50 and a TATA-like box centered around position -25. Mutational analyses indicated that both external promoter elements are important for efficient transcription. In addition, two sequence motifs can be identified in Hs7SL-1 and Hs7SL-3 at positions 10-19 and 50-60, respectively, downstream of the transcription start site that resemble putative A and B boxes. Single and multiple nucleotide substitutions in these regions also influenced transcription activity to a great extent. The requirement of intragenic functional A and B boxes in combination with the external ATF/CRE and TATA-like promoter elements for the efficient transcription of human 7SL RNA genes is reminiscent of at least two other classes of pol III-transcribed genes in human cells, such as Epstein-Barr virus-encoded EBER and vault RNA genes.
Asunto(s)
ARN Polimerasa III/metabolismo , ARN Citoplasmático Pequeño/genética , Partícula de Reconocimiento de Señal/genética , Transcripción Genética , Región de Flanqueo 5' , Secuencia de Bases , Sitios de Unión , Extractos Celulares , Cromosomas Humanos Par 14 , Clonación Molecular , Secuencia Conservada , Análisis Mutacional de ADN , ADN Complementario , Bases de Datos Genéticas , Amplificación de Genes , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Unión Proteica , TATA Box , Moldes GenéticosRESUMEN
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a ß-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.
Asunto(s)
Proteínas Arqueales , Haloferax volcanii , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN de Archaea , ARN de Transferencia , Ubiquitinas , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Uridina/genética , Uridina/metabolismoRESUMEN
Class I and II aminoacyl-tRNA synthetases (AARSs) attach amino acids to the 2'- and 3'-OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl-tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2'-OH. Here we show that two Class II AARSs, O-phosphoseryl- (SepRS) and pyrrolysyl-tRNA (PylRS) synthetases, aminoacylate the 2'- and 3'-OH, respectively. Structure-based-phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2'-OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.