Alanine tRNA Research Articles

Impaired 2',3'-cyclic phosphate tRNA repair causes thermo-sensitive genic male sterility in rice.

Hybrid rice, widely planted in Asia, is pathogen resistant and has superior yields, making it a major contributor to global food security. The two-line hybrid rice system, which utilizes mutants exhibiting photo-/thermo-sensitive genic male sterility (P/TGMS), is the leading hybrid rice breeding technology. Mutations in THERMO-SENSITIVE GENIC MALE STERILE 5 (TMS5) accounts for over 95% of current TGMS lines. We previously found that tms5 carries a mutation in ribonuclease ZS1. Despite its importance for breeding robust rice lines, the mechanism underlying tms5-mediated TGMS remains elusive. Here, we demonstrate that TMS5 is a tRNA 2',3'-cyclic phosphatase. The tms5 mutation leads to accumulation of 2',3'-cyclic phosphate (cP)-ΔCCA-tRNAs (tRNAs without 3'CCA ended with cP), which is exacerbated by high temperatures, and reduction in the abundance of mature tRNAs, particularly alanine tRNAs (tRNA-Alas). Overexpression of tRNA-Alas in the tms5 mutant restores male fertility to 70%. Remarkably, male fertility of tms5 mutant is completely restored at high temperatures by knocking out OsVms1 which encodes the enzyme for cP-ΔCCA-tRNA generation. Our study reveals the mechanism underlying tms5-mediated TGMS in rice and provides mechanistic insight into the further improvement of TGMS in hybrid crop development.

Carbon fixation via volatile fatty acids recovery from sewage sludge through electrochemical–pretreatment–based anaerobic digestion

Capturing the carbon in volatile fatty acids (VFA) produced from the anaerobic digestion (AD) of sewage sludge has the potential to not only provide economic benefits but also reduce greenhouse gas production. This study demonstrates a chemical–free method to collect VFA from an AD instead of methane that involves electrochemical pretreatment (EPT) of sludge. Experimental results show that applying 15 V EPT for 45 min enhances acidogenesis and selectively inhibits methanogenesis, leading to a substantial VFA accumulation (2563.1 ± 307.9 mg COD/L) and achieving 2.5 times more carbon fixation than via methane production. Interfacial thermodynamic analysis shows that EPT induces a decrease in both the repulsive electrostatic energy (from 152.9 kT to 12.2 kT) and the energy barrier (from 57.0 kT to 2.6 kT) in the sludge, leading to increased sludge aggregation and entrapment of microorganisms. Molecular docking sheds lights on how the methanogens interacts with the organic matter released from EPT (e.g., alanine–tRNA ligase), showing that these interactions potentially interfere with the proteins that are associated with the activities of the methanogens and the electron transfer pathways, thereby impeding methanogenesis. Integrating EPT into AD therefore facilitates the recovery of valuable VFA and the capture of carbon from freshwater sludge, providing notable economic and environmental benefits in sewage sludge treatment.

The Arabidopsis tDR Ala forms G-quadruplex structures that can be unwound by the DExH1 DEA(D/H)-box RNA helicase.

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.

Microbe-Responsive Proteomes During Plant–Microbe Interactions Between Rice Genotypes and the Multifunctional Methylobacterium oryzae CBMB20

BackgroundRice is colonized by plant growth promoting bacteria such as Methylobacterium leading to mutually beneficial plant–microbe interactions. As modulators of the rice developmental process, Methylobacterium influences seed germination, growth, health, and development. However, little is known about the complex molecular responsive mechanisms modulating microbe-driven rice development. The application of proteomics to rice-microbe interactions helps us elucidate dynamic proteomic responses mediating this association.ResultsIn this study, a total of 3908 proteins were detected across all treatments of which the non-inoculated IR29 and FL478 share up to 88% similar proteins. However, intrinsic differences appear in IR29 and FL478 as evident in the differentially abundant proteins (DAPs) and their associated gene ontology terms (GO). Successful colonization of M. oryzae CBMB20 in rice resulted to dynamic shifts in proteomes of both IR29 and FL478. The GO terms of DAPs for biological process in IR29 shifts in abundance from response to stimulus, cellular amino acid metabolic process, regulation of biological process and translation to cofactor metabolic process (6.31%), translation (5.41%) and photosynthesis (5.41%). FL478 showed a different shift from translation-related to response to stimulus (9%) and organic acid metabolic acid (8%). Both rice genotypes also showed a diversification of GO terms due to the inoculation of M. oryzae CBMB20. Specific proteins such as peptidyl-prolyl cis–trans isomerase (A2WJU9), thiamine thiazole synthase (A2YM28), and alanine—tRNA ligase (B8B4H5) upregulated in IR29 and FL478 indicate key mechanisms of M. oryzae CBMB20 mediated plant growth promotion in rice.ConclusionsInteraction of Methylobacterium oryzae CBMB20 to rice results in a dynamic, similar, and plant genotype-specific proteomic changes supporting associated growth and development. The multifaceted CBMB20 expands the gene ontology terms and increases the abundance of proteins associated with photosynthesis, diverse metabolic processes, protein synthesis and cell differentiation and fate potentially attributed to the growth and development of the host plant. The specific proteins and their functional relevance help us understand how CBMB20 mediate growth and development in their host under normal conditions and potentially link subsequent responses when the host plants are exposed to biotic and abiotic stresses.

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

Alanine tRNAs Translate Environment Into Behavior in Caenorhabditis elegans.

Caenorhabditis elegans nematodes produce and maintain imprints of attractive chemosensory cues to which they are exposed early in life. Early odor-exposure increases adult chemo-attraction to the same cues. Imprinting is transiently or stably inherited, depending on the number of exposed generations. We show here that the Alanine tRNA (UGC) plays a central role in regulating C. elegans chemo-attraction. Naive worms fed on tRNAAla (UGC) purified from odor-experienced worms, acquire odor-specific imprints. Chemo-attractive responses require the tRNA-modifying Elongator complex sub-units 1 (elpc-1) and 3 (elpc-3) genes. elpc-3 deletions impair chemo-attraction, which is fully restored by wild-type tRNAAla (UGC) feeding. A stably inherited decrease of odor-specific responses ensues from early odor-exposition of elpc-1 deletion mutants. tRNAAla (UGC) may adopt various chemical forms to mediate the cross-talk between innately-programmed and environment-directed chemo-attractive behavior.

Methyltransferase-like 21C (METTL21C) methylates alanine tRNA synthetase at Lys-943 in muscle tissue

Protein-lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70-kDa protein 8 (HSPA8). However, a clear in vitro methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate(s) in vivo is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine tRNA synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both in vitro and in vivoIn vitro METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells depends upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a bona fide in vitro substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.

Transfection of Small Noncoding RNAs into Arabidopsis thaliana Protoplasts.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.

Synthetic biology in China: Review and prospects

Synthetic biology is an emerging science and a typical convergence technology. On the basis of system biology, integrating the principles of engineering science and adopting bottom-up strategies, it aims to reveal the mechanism of life and to build a new generation of biotechnology, through designing and constructing new biological systems or redesigning the existing biological systems, which is therefore summarized in Chinese as “build-to-learn” and “build-to-use”. In early years, Chinese scientists were the first to artificially synthesize the functional protein (bovine insulin) and ribonucleic acid (yeast alanine tRNA). In recent years, they have been engaged in chromosomal synthesis and chromosomal engineering, genome editing, biological chassis construction, quantitative engineering biology, biological parts and gene circuits engineering, synthetic metabolism of natural products and organic chemical products, and enabling technologies (e.g. genome editing and computer simulation), and made a series of important discoveries and innovative achievements. These have given Chinese scientists an important role in the international consortium of synthetic biology. By the time to review 70 years’ development of science and technology in China, this article is to commemorate the pioneers and inspire the early career academics in the field, meanwhile discuss experiences and lessons, and identify future priorities and opportunities. Nowadays, synthetic biology is booming in the world, and Chinese scientists will be delighted to play vital role in it.

Mitochondrial tRNAAla 5601C>T variant may affect the clinical expression of the LHON‑related ND4 11778G>A mutation in a family.

Certain mutations in mitochondrial DNA (mtDNA) are associated with Leber's hereditary optic neuropathy (LHON). In particular, the well-known NADH dehydrogenase 4 (ND4) m.11778G>A mutation is one of the most common LHON-associated primary mutations worldwide. However, how specific mtDNA mutations, or variants, affect LHON penetrance is not fully understood. The aim of the current study was to explore the relationship between mtDNA mutations and LHON, and to provide useful information for early detection and prevention of this disease. Following the molecular characterization of a Han Chinese family with maternally inherited LHON, four out of eight matrilineal relatives demonstrated varying degrees of both visual impairment and age of onset. Through PCR amplification of mitochondrial genomes and direct Sanger sequencing analysis, a homoplasmic mitochondrial-encoded ND4 m.11778G>A mutation, alongside a set of genetic variations belonging to human mtDNA haplogroup B5b1 were identified. Among these sequence variants, alanine transfer RNA (tRNA)Ala m.5601C>T was of particular interest. This variant occurred at position 59 in the TψC loop and altered the base pairing, which led to mitochondrial RNA (mt-RNA) metabolism failure and defects in mitochondrial protein synthesis. Bioinformatics analysis suggested that the m.5601C>T variant altered tRNAAla structure. Therefore, impaired mitochondrial functions caused by the ND4 m.11778G>A mutation may be enhanced by the mt-tRNAAla m.5601C>T variant. These findings suggested that the tRNAAla m.5601C>T variant might modulate the clinical manifestation of the LHON-associated primary mutation.

Rapid Genetic Code Evolution in Green Algal Mitochondrial Genomes.

Genetic code deviations involving stop codons have been previously reported in mitochondrial genomes of several green plants (Viridiplantae), most notably chlorophyte algae (Chlorophyta). However, as changes in codon recognition from one amino acid to another are more difficult to infer, such changes might have gone unnoticed in particular lineages with high evolutionary rates that are otherwise prone to codon reassignments. To gain further insight into the evolution of the mitochondrial genetic code in green plants, we have conducted an in-depth study across mtDNAs from 51 green plants (32 chlorophytes and 19 streptophytes). Besides confirming known stop-to-sense reassignments, our study documents the first cases of sense-to-sense codon reassignments in Chlorophyta mtDNAs. In several Sphaeropleales, we report the decoding of AGG codons (normally arginine) as alanine, by tRNA(CCU) of various origins that carry the recognition signature for alanine tRNA synthetase. In Chromochloris, we identify tRNA variants decoding AGG as methionine and the synonymous codon CGG as leucine. Finally, we find strong evidence supporting the decoding of AUA codons (normally isoleucine) as methionine in Pycnococcus. Our results rely on a recently developed conceptual framework (CoreTracker) that predicts codon reassignments based on the disparity between DNA sequence (codons) and the derived protein sequence. These predictions are then validated by an evaluation of tRNA phylogeny, to identify the evolution of new tRNAs via gene duplication and loss, and structural modifications that lead to the assignment of new tRNA identities and a change in the genetic code.

Alanine tRNA Translate Environment into Behavior in <i>Caenorhabditis Elegans</i>

Caenorhabditis elegans nematodes produce and keep imprints of attractive chemosensory cues to which they are exposed early in life. These imprints enhance adult chemo-attraction to the same cues. Depending on the number of odor-exposed generations, imprinting is transiently or stably inherited. Strinkingly, we have found that early odor-exposed C. elegans produce odor-specific forms of the transfer RNAAla (UGC). Naive animals fed on these tRNAs acquire transient or stable odor-specific imprinting. The tRNAAla (UGC) controls C. elegans chemo-attractive responses through the multifunctional Elongator complex. Mutations that affect the functions of Elongator sub-units 1 or 3, either impaired chemo-attraction, or definitely abolish responses to the odors nematodes were exposed. We hypothesize that early olfactory experiences translate into tRNAAla (UGC) bearing odor-specific signatures. These diffusible odor memory, together with Elongator, can stably reprogram the C. elegans chemo-attractive behavior.

The Proposal of Series Projects about Artificial Synthetic Life(1965—1968)

After the total synthesis of crystalline bovine insulin, the research team members, the experts at the appraisal meeting, and the leaders of Ministry of Higher Education and Chinese Academy of Sciences proposed some research plans. But these plans were suspended because of the broken of the Culture Revolution. A year later, the second Beijing science seminar was scheduled to be held in the summer of 1968.It prompted these plans to start again. According to vice Premier Nie Rongzhen’s instructions and the August 24, 1964 night talks of Mao Zedong, the relevant personnel held a series of conferences in 1967—1968, and proposed series projects about artificial synthetic life, such as the determination of crystal structure of insulin, the synthesis of tobacco mosaic virus protein subunit, the synthesis of yeast alanine tRNA.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease

SummaryMutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes.

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including “codon capture,” “genome streamlining,” and “ambiguous intermediate” theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNAAla containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.

Data on true tRNA diversity among uncultured and bacterial strains.

Complete genome sequences of two uncultured archaea (BX649197 and CR937008) and 10 uncultured bacteria (AC160099, FP245538-FP245540, FP312972, FP312974-75, FP312977, FP312985 and NZ_JPJG01000067) were used for creation of digital data of tRNA. tRNAscan-SE and ENDMEMO GC calculating tools were used for detection of tRNA, drawing their structures and calculation of GC percent. Seven archaeal and 48 bacterial tRNA were detected from above 12 sequences. Four archaeal and 30 bacterial tRNA showed cove score more than 20% are called as true tRNA. Three tRNA of uncultured bacteria (AC160099) has the presence of the variable loop. The tRNA of FP245540, FP245575, FP245577 and FP245585 has one variable loop each. The true tRNA of archaea were Alanine, Arginine and Cysteine-type tRNA, while the majority of bacteria true tRNA classified as Alanine, Glutamic acid, Isoleucine, Leucine, Methionine, Phenylalanine, Proline and Valine-type tRNA with cove score ranged from 70% to 97.15%. Archaeal and bacterial have GC content approximately 43% and 34.7–63.3% respectively. Archaeal tRNA has 60.4–64.2% GC content. Similarly, bacterial tRNA contributed 49.3–66.3% GC content to the total GC content. This generated data is useful for studies on diversity of tRNA among prokaryotes.

PNRD: a plant non-coding RNA database.

The first ncRNA found was an alanine tRNA in baker's yeast, and the first detected microRNAs (miRNAs) promoted ncRNA research to a whole new level. Research on ncRNAs in animals has focused on the medical field, while in plant scientists are more concerned with improving agronomic traits. In 2010, we constructed a plant miRNA database named PMRD to meet the demand for miRNA research in plants. To provide a way to do fundamental research on plant ncRNAs and take full advantage of tremendous public resources, we designed an updated platform called plant ncRNA database (PNRD) based on its predecessor PMRD, which is accessible at http://structuralbiology.cau.edu.cn/PNRD. We collected a total of 25739 entries of 11 different types of ncRNAs from 150 plant species. Targets of miRNAs were extended to 178138 pairs in 46 species, while the number of miRNA expression profiles reached 35. Improvements in PNRD are not only the larger amounts of data, but also better service, such as a more user-friendly interface, more multifunctional and browsing options and more background data for users to download. We also integrated currently prevalent technologies and toolkits to strengthen the capability of the database and provide a one-stop service for scientific users.

Optimization protein productivity of human interleukin-2 through codon usage, gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.

Structural biology: wobble puts RNA on target.

Enzymes that attach amino acids to transfer RNAs during protein synthesis must recognize both substrates specifically. Crystal structures reveal a mechanism that explains the RNA specificity for one such system. See Article p.507 tRNA synthetases must attach the appropriate amino acid to the appropriate, cognate tRNA to maintain proper translation of the genetic code into protein. A single non-Watson–Crick base pair in the tRNA for alanine, G3·U70, ensures correct aminoacylation by the synthetase (AlaRS). Shigeyuki Yokoyama and colleagues report two crystal structures of AlaRS from the archaeon Archaeoglobus fulgidus, in complexes with tRNAAla bearing G3·U70 (tRNAAla/GU) and with a variant bearing A3·U70 (tRNAAla/AU), together with an alanyl-AMP analogue. Comparison of the structures reveals the basis for selectivity. The unique geometry of G3·U70 orients the CCA region of tRNA and guides the terminal adenosine into the catalytic site. This mechanism explains how a small number of nucleotides distant from the catalytic site can determine the reactivity of specific aminoacylation.