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1.
RNA Biol ; 20(1): 791-804, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-37776539

RESUMEN

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.


Asunto(s)
Anticodón , ARN de Transferencia de Alanina , Anticodón/genética , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia/metabolismo , Codón , Alanina/genética , Alanina/metabolismo , Biosíntesis de Proteínas
2.
J Biol Chem ; 299(9): 105149, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37567477

RESUMEN

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.


Asunto(s)
Alanina-ARNt Ligasa , Caenorhabditis elegans , Motivos de Nucleótidos , ARN de Transferencia de Alanina , Animales , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Secuencia Conservada , Citoplasma/enzimología , ADN/química , ADN/metabolismo , Ligandos , Lisina/metabolismo , Mitocondrias/enzimología , Dominios Proteicos , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , Especificidad por Sustrato , Conformación de Ácido Nucleico
3.
Commun Biol ; 6(1): 314, 2023 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-36959394

RESUMEN

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology, consisting of catalytic, tRNA-recognition, editing, and C-Ala domains. The catalytic and tRNA-recognition domains catalyze aminoacylation, the editing domain hydrolyzes mischarged tRNAAla, and C-Ala-the major tRNA-binding module-targets the elbow of the L-shaped tRNAAla. Interestingly, a mini-AlaRS lacking the editing and C-Ala domains is recovered from the Tupanvirus of the amoeba Acanthamoeba castellanii. Here we show that Tupanvirus AlaRS (TuAlaRS) is phylogenetically related to its host's AlaRS. Despite lacking the conserved amino acid residues responsible for recognition of the identity element of tRNAAla (G3:U70), TuAlaRS still specifically recognized G3:U70-containing tRNAAla. In addition, despite lacking C-Ala, TuAlaRS robustly binds and charges microAla (an RNA substrate corresponding to the acceptor stem of tRNAAla) as well as tRNAAla, indicating that TuAlaRS exclusively targets the acceptor stem. Moreover, this mini-AlaRS could functionally substitute for yeast AlaRS in vivo. This study suggests that TuAlaRS has developed a new tRNA-binding mode to compensate for the loss of C-Ala.


Asunto(s)
Alanina-ARNt Ligasa , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Escherichia coli/genética , ARN de Transferencia/metabolismo
4.
Philos Trans R Soc Lond B Biol Sci ; 378(1871): 20220029, 2023 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-36633285

RESUMEN

By linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. We recently found that a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. Here, we hypothesized that human tRNA variants that misread glycine (Gly) codons with alanine (Ala) will also disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3 : U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions; however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.


Asunto(s)
Alanina-ARNt Ligasa , Biosíntesis de Proteínas , Humanos , Alanina/genética , Alanina/química , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Codón/genética , Glicina/genética , Glicina/metabolismo , Proteostasis , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Glicerina/metabolismo
5.
J Biol Chem ; 298(3): 101601, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35065077

RESUMEN

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species "sinks" to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.


Asunto(s)
Alanina-ARNt Ligasa , Escherichia coli , Alanina-ARNt Ligasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Estrés Oxidativo , Proteoma , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Especies Reactivas de Oxígeno/metabolismo
6.
J Biol Chem ; 297(1): 100816, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34023389

RESUMEN

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.


Asunto(s)
Mitocondrias/metabolismo , ARN de Transferencia de Alanina/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis , Secuencia de Bases , Citocromos c/metabolismo , Transporte de Electrón , Humanos , Potencial de la Membrana Mitocondrial , Proteínas Mitocondriales/metabolismo , Mitofagia , Mutación/genética , Conformación de Ácido Nucleico , Fosforilación Oxidativa , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genética , ARN Mitocondrial/genética , ARN de Transferencia de Alanina/química , Especies Reactivas de Oxígeno/metabolismo , Aminoacilación de ARN de Transferencia
7.
Mol Cell ; 81(1): 104-114.e6, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33259811

RESUMEN

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.


Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Extensión de la Cadena Peptídica de Translación , ARN Bacteriano/metabolismo , ARN de Transferencia de Alanina/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , ARN Bacteriano/genética , ARN de Transferencia de Alanina/genética
8.
RNA ; 27(1): 66-79, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33023933

RESUMEN

Most mammalian cytoplasmic tRNAs contain ribothymidine (T) and pseudouridine (Ψ) at positions 54 and 55, respectively. However, some tRNAs contain Ψ at both positions. Several Ψ54-containing tRNAs function as primers in retroviral DNA synthesis. The Ψ54 of these tRNAs is produced by PUS10, which can also synthesize Ψ55. Two other enzymes, TRUB1 and TRUB2, can also produce Ψ55. By nearest-neighbor analyses of tRNAs treated with recombinant proteins and subcellular extracts of wild-type and specific Ψ55 synthase knockdown cells, we determined that while TRUB1, PUS10, and TRUB2 all have tRNA Ψ55 synthase activities, they have different tRNA structural requirements. Moreover, these activities are primarily present in the nucleus, cytoplasm, and mitochondria, respectively, suggesting a compartmentalization of Ψ55 synthase activity. TRUB1 produces the Ψ55 of most elongator tRNAs, but cytoplasmic PUS10 produces both Ψs of the tRNAs with Ψ54Ψ55. The nuclear isoform of PUS10 is catalytically inactive and specifically binds the unmodified U54U55 versions of Ψ54Ψ55-containing tRNAs, as well as the A54U55-containing tRNAiMet This binding inhibits TRUB1-mediated U55 to Ψ55 conversion in the nucleus. Consequently, the U54U55 of Ψ54Ψ55-containing tRNAs are modified by the cytoplasmic PUS10. Nuclear PUS10 does not bind the U55 versions of T54Ψ55- and A54Ψ55-containing elongator tRNAs. Therefore, TRUB1 is able to produce Ψ55 in these tRNAs. In summary, the tRNA Ψ55 synthase activities of TRUB1 and PUS10 are not redundant but rather are compartmentalized and act on different sets of tRNAs. The significance of this compartmentalization needs further study.


Asunto(s)
Núcleo Celular/genética , Citoplasma/genética , Hidroliasas/genética , Mitocondrias/genética , Seudouridina/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Triptófano/genética , Animales , Sitios de Unión , Compartimento Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Células HEK293 , Humanos , Hidroliasas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocondrias/metabolismo , Células PC-3 , Unión Proteica , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Metionina/metabolismo , ARN de Transferencia de Triptófano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera
9.
RNA ; 26(11): 1519-1529, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32737189

RESUMEN

tRNA molecules have well-defined sequence conservations that reflect the conserved tertiary pairs maintaining their architecture and functions during the translation processes. An analysis of aligned tRNA sequences present in the GtRNAdb database (the Lowe Laboratory, University of California, Santa Cruz) led to surprising conservations on some cytosolic tRNAs specific for alanine compared to other tRNA species, including tRNAs specific for glycine. First, besides the well-known G3oU70 base pair in the amino acid stem, there is the frequent occurrence of a second wobble pair at G30oU40, a pair generally observed as a Watson-Crick pair throughout phylogeny. Second, the tertiary pair R15/Y48 occurs as a purine-purine R15/A48 pair. Finally, the conserved T54/A58 pair maintaining the fold of the T-loop is observed as a purine-purine A54/A58 pair. The R15/A48 and A54/A58 pairs always occur together. The G30oU40 pair occurs alone or together with these other two pairs. The pairing variations are observed to a variable extent depending on phylogeny. Among eukaryotes, insects display all variations simultaneously, whereas mammals present either the G30oU40 pair or both R15/A48 and A54/A58. tRNAs with the anticodon 34A(I)GC36 are the most prone to display all those pair variations in mammals and insects. tRNAs with anticodon Y34GC36 have preferentially G30oU40 only. These unusual pairs are not observed in bacterial, nor archaeal, tRNAs, probably because of the avoidance of A34-containing anticodons in four-codon boxes. Among eukaryotes, these unusual pairing features were not observed in fungi and nematodes. These unusual structural features may affect, besides aminoacylation, transcription rates (e.g., 54/58) or ribosomal translocation (30/40).


Asunto(s)
Insectos/genética , Mamíferos/genética , ARN de Transferencia de Alanina/química , Animales , Secuencia de Bases , Secuencia Conservada , Bases de Datos Genéticas , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Filogenia , Pliegue del ARN , ARN de Transferencia de Alanina/metabolismo
10.
Biosystems ; 197: 104206, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32640271

RESUMEN

The unique G3:U70 base pair in the acceptor stem of tRNAAla has been shown to be a critical recognition site by alanyl-tRNA synthetase (AlaRS). The base pair resides on one of the arms of the L-shaped structure of tRNA (minihelix) and the genetic code has likely evolved from a primordial tRNA-aaRS (aminoacyl-tRNA synthetase) system. In terms of the evolution of tRNA, incorporation of a G:U base pair in the structure would be important. Here, we found that two independent short hairpin RNAs change their conformation through kissing-loop interactions, finally forming a minihelix-like structure, in which the G3:U70 base pair is incorporated. The RNA system can be properly aminoacylated by the minimal Escherichia coli AlaRS variant with alanylation activity (AlaRS442N). Thus, characteristic structural features produced via kissing-loop interactions may provide important clues into the evolution of RNA.


Asunto(s)
Aminoacilación/genética , Evolución Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/genética , ARN de Transferencia de Alanina/genética , Alanina-ARNt Ligasa , Aminoacil-ARNt Sintetasas , Emparejamiento Base , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Modelos Moleculares , Pliegue del ARN , ARN Interferente Pequeño/metabolismo , ARN de Transferencia de Alanina/metabolismo
11.
RNA ; 25(5): 607-619, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30737359

RESUMEN

Adenosine deaminase acting on transfer RNA (ADAT) is an essential eukaryotic enzyme that catalyzes the deamination of adenosine to inosine at the first position of tRNA anticodons. Mammalian ADATs modify eight different tRNAs, having increased their substrate range from a bacterial ancestor that likely deaminated exclusively tRNAArg Here we investigate the recognition mechanisms of tRNAArg and tRNAAla by human ADAT to shed light on the process of substrate expansion that took place during the evolution of the enzyme. We show that tRNA recognition by human ADAT does not depend on conserved identity elements, but on the overall structural features of tRNA. We find that ancestral-like interactions are conserved for tRNAArg, while eukaryote-specific substrates use alternative mechanisms. These recognition studies show that human ADAT can be inhibited by tRNA fragments in vitro, including naturally occurring fragments involved in important regulatory pathways.


Asunto(s)
Adenosina Desaminasa/metabolismo , Anticodón/química , ARN de Transferencia de Alanina/química , ARN de Transferencia de Arginina/química , Adenosina/metabolismo , Adenosina Desaminasa/genética , Anticodón/genética , Anticodón/metabolismo , Secuencia de Bases , Desaminación , Evolución Molecular , Expresión Génica , Humanos , Inosina/metabolismo , Conformación de Ácido Nucleico , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismo , Alineación de Secuencia , Especificidad por Sustrato
12.
Elife ; 72018 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-30091703

RESUMEN

D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNAGly misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3•U70 for tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.


Asunto(s)
Aminoaciltransferasas/metabolismo , Escherichia coli/metabolismo , Glicina/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Glicerina/metabolismo , Animales , Escherichia coli/enzimología , Ratones
13.
Nat Commun ; 9(1): 1887, 2018 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-29760453

RESUMEN

The genetic code used in nuclear genes is almost universal, but here we report that it changed three times in parallel during the evolution of budding yeasts. All three changes were reassignments of the codon CUG, which is translated as serine (in 2 yeast clades), alanine (1 clade), or the 'universal' leucine (2 clades). The newly discovered Ser2 clade is in the final stages of a genetic code transition. Most species in this clade have genes for both a novel tRNASer(CAG) and an ancestral tRNALeu(CAG) to read CUG, but only tRNASer(CAG) is used in standard growth conditions. The coexistence of these alloacceptor tRNA genes indicates that the genetic code transition occurred via an ambiguous translation phase. We propose that the three parallel reassignments of CUG were not driven by natural selection in favor of their effects on the proteome, but by selection to eliminate the ancestral tRNALeu(CAG).


Asunto(s)
Codón , Código Genético , Genoma Fúngico , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Serina/genética , Saccharomycetales/genética , Alanina/genética , Alanina/metabolismo , Evolución Molecular , Leucina/genética , Leucina/metabolismo , Conformación de Ácido Nucleico , Filogenia , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Serina/metabolismo , Saccharomycetales/clasificación , Saccharomycetales/metabolismo , Selección Genética , Serina/genética , Serina/metabolismo
14.
RNA Biol ; 15(4-5): 492-499, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29168417

RESUMEN

Horizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans encodes an integrative-conjugative genetic element (ICEAfe1) inserted in the gene encoding a tRNAAla. This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICEAcaTY.2) is also found in an equivalent position in the genome of Acidithiobacillus caldus. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and in vivo integrations assays in the heterologous model Escherichia coli, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target attB sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of Acidithiobacillus genus.


Asunto(s)
Acidithiobacillus/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Transferencia de Gen Horizontal , ARN de Transferencia de Alanina/genética , Acidithiobacillus/metabolismo , Anticodón/química , Anticodón/metabolismo , Sitios de Ligazón Microbiológica , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Bacterianos/química , Cromosomas Bacterianos/metabolismo , Daño del ADN , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Integrasas/genética , Integrasas/metabolismo , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , ARN de Transferencia de Alanina/metabolismo , Recombinación Genética , Sintenía
15.
Sci Rep ; 7(1): 6709, 2017 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-28751745

RESUMEN

Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2'OH → 2'H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNAAlaugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2'OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2'H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAlaugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAlaugc and the translocation of the P-site deacylated tRNAAlaugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2'OH groups in tRNAs in translation.


Asunto(s)
Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , ARN de Transferencia de Alanina/química , Ribosomas/genética , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura
16.
Biochemistry ; 56(31): 4029-4038, 2017 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-28703578

RESUMEN

Transfer RNAs (tRNAs) are among the most heavily modified RNA species. Posttranscriptional tRNA modifications (ptRMs) play fundamental roles in modulating tRNA structure and function and are being increasingly linked to human physiology and disease. Detection of ptRMs is often challenging, expensive, and laborious. Restriction fragment length polymorphism (RFLP) analyses study the patterns of DNA cleavage after restriction enzyme treatment and have been used for the qualitative detection of modified bases on mRNAs. It is known that some ptRMs induce specific and reproducible base "mutations" when tRNAs are reverse transcribed. For example, inosine, which derives from the deamination of adenosine, is detected as a guanosine when an inosine-containing tRNA is reverse transcribed, amplified via polymerase chain reaction (PCR), and sequenced. ptRM-dependent base changes on reverse transcription PCR amplicons generated as a consequence of the reverse transcription reaction might create or abolish endonuclease restriction sites. The suitability of RFLP for the detection and/or quantification of ptRMs has not been studied thus far. Here we show that different ptRMs can be detected at specific sites of different tRNA types by RFLP. For the examples studied, we show that this approach can reliably estimate the modification status of the sample, a feature that can be useful in the study of the regulatory role of tRNA modifications in gene expression.


Asunto(s)
Adenosina Desaminasa/metabolismo , Modelos Biológicos , Polimorfismo de Longitud del Fragmento de Restricción , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Treonina/metabolismo , Adenosina/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Emparejamiento Base , Biología Computacional , Desaminación , Sistemas Especialistas , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Inosina/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN de Transferencia de Alanina/antagonistas & inhibidores , ARN de Transferencia de Treonina/antagonistas & inhibidores , ARN de Transferencia de Valina/antagonistas & inhibidores , ARN de Transferencia de Valina/metabolismo , Transcripción Reversa , Especificidad por Sustrato
17.
Proc Natl Acad Sci U S A ; 112(34): 10691-6, 2015 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-26261323

RESUMEN

The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.


Asunto(s)
Aminoaciltransferasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Fosfatidilgliceroles/metabolismo , Pseudomonas aeruginosa/enzimología , Factores R , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Lisina/metabolismo , Aminoacilación , Aminoaciltransferasas/metabolismo , Bacillus/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/biosíntesis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Fosfatidilgliceroles/biosíntesis , Conformación Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad , Especificidad por Sustrato
18.
Science ; 347(6217): 75-8, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25554787

RESUMEN

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").


Asunto(s)
Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Microscopía por Crioelectrón , Conformación de Ácido Nucleico , Conformación Proteica , ARN Mensajero/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Treonina/química , ARN de Transferencia de Treonina/metabolismo , Proteínas de Unión al ARN , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Ubiquitina-Proteína Ligasas/ultraestructura
19.
Nucleic Acids Res ; 42(15): 10061-72, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25056309

RESUMEN

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5' nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA(Gln) were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.


Asunto(s)
Codón de Terminación , Terminación de la Cadena Péptídica Traduccional , ARN de Transferencia de Glutamina/metabolismo , Saccharomyces cerevisiae/genética , Aminoácidos/metabolismo , Anticodón , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , ARN de Transferencia de Alanina/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Nucleic Acids Res ; 42(1): 499-508, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24049072

RESUMEN

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted tRNA(Ala)UAG is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that tRNA(Ala)UAG is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNA(Ala); a G3A change in Ashbya tRNA(Ala)UAG abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae tRNA(Thr)UAG confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.


Asunto(s)
Codón , Eremothecium/genética , Mitocondrias/genética , ARN de Transferencia de Alanina/metabolismo , Alanina/metabolismo , Alanina-ARNt Ligasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Eremothecium/enzimología , Leucina/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , ARN de Transferencia/química , ARN de Transferencia/metabolismo , ARN de Transferencia de Alanina/química
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