Using selenocysteine-specific reporters to screen for efficient tRNASec variants.

The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.

Female Mice with Selenocysteine tRNA Deletion in Agrp Neurons Maintain Leptin Sensitivity and Resist Weight Gain While on a High-Fat Diet.

The role of the essential trace element selenium in hypothalamic physiology has begun to come to light over recent years. Selenium is used to synthesize a family of proteins participating in redox reactions called selenoproteins, which contain a selenocysteine residue in place of a cysteine. Past studies have shown that disrupted selenoprotein expression in the hypothalamus can adversely impact energy homeostasis. There is also evidence that selenium supports leptin signaling in the hypothalamus by maintaining proper redox balance. In this study, we generated mice with conditional knockout of the selenocysteine tRNA[Ser]Sec gene (Trsp) in an orexigenic cell population called agouti-related peptide (Agrp)-positive neurons. We found that female TrspAgrpKO mice gain less weight while on a high-fat diet, which occurs due to changes in adipose tissue activity. Female TrspAgrpKO mice also retained hypothalamic sensitivity to leptin administration. Male mice were unaffected, however, highlighting the sexually dimorphic influence of selenium on neurobiology and energy homeostasis. These findings provide novel insight into the role of selenoproteins within a small yet heavily influential population of hypothalamic neurons.

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions.

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNASec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNASec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNASec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNASec. Subsequently, SelB.GTP.tRNASec recognizes the mRNA SECIS to deliver the tRNASec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNASec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.

Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria.

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

Historical Roles of Selenium and Selenoproteins in Health and Development: The Good, the Bad and the Ugly.

Selenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found to be an essential element in the diet of humans, all other mammals, and many other life forms. It has many health benefits that include, for example, roles in preventing heart disease and certain forms of cancer, slowing AIDS progression in HIV patients, supporting male reproduction, inhibiting viral expression, and boosting the immune system, and it also plays essential roles in mammalian development. Elucidating the molecular biology of selenium over the past 40 years generated an entirely new field of science which encompassed the many novel features of selenium. These features were (1) how this element makes its way into protein as the 21st amino acid in the genetic code, selenocysteine (Sec); (2) the vast amount of machinery dedicated to synthesizing Sec uniquely on its tRNA; (3) the incorporation of Sec into protein; and (4) the roles of the resulting Sec-containing proteins (selenoproteins) in health and development. One of the research areas receiving the most attention regarding selenium in health has been its role in cancer prevention, but further research has also exposed the role of this element as a facilitator of various maladies, including cancer.

Seryl-tRNA synthetase specificity for tRNASec in Bacterial Sec biosynthesis.

tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.

A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 µg/mL) beyond that of the three-kinase system (∼400 µg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.

Cold acclimation triggers lipidomic and metabolic adjustments in the spotted wing drosophila Drosophila suzukii (Matsumara).

Chronic cold exposure is detrimental to chill susceptible insects that may accumulate chill injuries. To cope with deleterious effects of cold temperature, insects employ a variety of physiological strategies and metabolic adjustments, such as production of cryoprotectants, or remodeling of cellular membranes. Cold tolerance is a key element determining the fundamental niche of species. Because Drosophila suzukii is an invasive fruit pest, originating from East Asia, knowledge about its thermal biology is urgently needed. Physiological mechanisms underlying cold tolerance plasticity remain poorly understood in this species. Here, we explored metabolic and lipidomic modifications associated with the acquisition of cold tolerance in D. suzukii using Omics technologies (LC- and GC-MS/MS). In both cold-acclimated males and females, we observed physiological changes consistent with homeoviscous/homeophasic adaptation of membranes: reshuffling of phospholipid head groups and increasing unsaturation rate of fatty acids. Modification of fatty acids unsaturation were also observed in triacylglycerides, which would likely increase accessibility of lipid reserves. At the metabolic level, we observed clear-cut differentiation of metabolic profiles with cold-acclimated metabotypes showing accumulation of several potential cryoprotectants (sugars and amino acids). Metabolic pathway analyses indicated a remodeling of various processes, including purine metabolism and aminoacyl tRNA biosynthesis. These data provide a large-scale characterization of lipid rearrangements and metabolic pathway modifications in D. suzukii in response to cold acclimation and contribute to characterizing the strategies used by this species to modulate cold tolerance.

Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs.

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNASec , which is subsequently converted by selenocysteine synthase to Sec-tRNASec . Escherichia coli SerRS serylates tRNASec poorly; this may hinder efficient production of designer selenoproteins in vivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNASec synthesis. They display 10-, 8-, and 4-fold increased kcat /KM values compared to wild-type SerRS using synthetic tRNASec species as substrates. The enzyme variants also facilitate in vivo read-through of a UAG codon in the position of the critical serine146 of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.

Methanomethylophilus alvus Mx1201 Provides Basis for Mutual Orthogonal Pyrrolysyl tRNA/Aminoacyl-tRNA Synthetase Pairs in Mammalian Cells.

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded noncanonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei ( Mma PylRS/PylT) being the most active and versatile to date. We found a pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that this PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its noncognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

Coding of Class I and II Aminoacyl-tRNA Synthetases.

The aminoacyl-tRNA synthetases and their cognate transfer RNAs translate the universal genetic code. The twenty canonical amino acids are sufficiently diverse to create a selective advantage for dividing amino acid activation between two distinct, apparently unrelated superfamilies of synthetases, Class I amino acids being generally larger and less polar, Class II amino acids smaller and more polar. Biochemical, bioinformatic, and protein engineering experiments support the hypothesis that the two Classes descended from opposite strands of the same ancestral gene. Parallel experimental deconstructions of Class I and II synthetases reveal parallel losses in catalytic proficiency at two novel modular levels-protozymes and Urzymes-associated with the evolution of catalytic activity. Bi-directional coding supports an important unification of the proteome; affords a genetic relatedness metric-middle base-pairing frequencies in sense/antisense alignments-that probes more deeply into the evolutionary history of translation than do single multiple sequence alignments; and has facilitated the analysis of hitherto unknown coding relationships in tRNA sequences. Reconstruction of native synthetases by modular thermodynamic cycles facilitated by domain engineering emphasizes the subtlety associated with achieving high specificity, shedding new light on allosteric relationships in contemporary synthetases. Synthetase Urzyme structural biology suggests that they are catalytically-active molten globules, broadening the potential manifold of polypeptide catalysts accessible to primitive genetic coding and motivating revisions of the origins of catalysis. Finally, bi-directional genetic coding of some of the oldest genes in the proteome places major limitations on the likelihood that any RNA World preceded the origins of coded proteins.

Insights into substrate promiscuity of human seryl-tRNA synthetase.

Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNASer) and the noncognate selenocysteine tRNA (tRNASec). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNASer, tRNASec, and 10 mutant and chimeric constructs in which elements of tRNASer were transposed onto tRNASec We show that human SerRS only subtly prefers tRNASer to tRNASec, and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNASec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNASec and tRNASer dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNASec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis.

Ribosome dynamics during decoding.

Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNASec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation.This article is part of the themed issue 'Perspectives on the ribosome'.

Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress.

The relationship between loss of hypothalamic function and onset of diabetes mellitus remains elusive. Therefore, we generated a targeted oxidative-stress murine model utilizing conditional knockout (KO) of selenocysteine-tRNA (Trsp) using rat-insulin-promoter-driven-Cre (RIP-Cre). These Trsp-KO (TrspRIPKO) mice exhibit deletion of Trsp in both hypothalamic cells and pancreatic ß cells, leading to increased hypothalamic oxidative stress and severe insulin resistance. Leptin signals are suppressed, and numbers of proopiomelanocortin-positive neurons in the hypothalamus are decreased. In contrast, Trsp-KO mice (TrspIns1KO) expressing Cre specifically in pancreatic ß cells, but not in the hypothalamus, do not display insulin and leptin resistance, demonstrating a critical role of the hypothalamus in the onset of diabetes mellitus. Nrf2 (NF-E2-related factor 2) regulates antioxidant gene expression. Increased Nrf2 signaling suppresses hypothalamic oxidative stress and improves insulin and leptin resistance in TrspRIPKO mice. Thus, Nrf2 harbors the potential to prevent the onset of diabetic mellitus by reducing hypothalamic oxidative damage.

A new set of assays for the discovery of aminoacyl-tRNA synthetase inhibitors.

Current biochemical methods available to monitor the activity of aminoacyl-tRNA synthetases (ARS) are ill-suited to high-throughput screening approaches for the identification of small-molecule inhibitors of these enzymes. In an attempt to improve the limitations of current assays we have developed a suite of new methods designed to streamline the discovery of new ARS antagonists. This set of assays includes approaches to monitor ARS activity in vitro, in human cells, and in bacteria. They are applicable to several ARSs from any given organism, can be easily adapted to very high-throughput set-ups, and allow for a multi-factorial selection of drug candidates.

The RNA-binding protein Secisbp2 differentially modulates UGA codon reassignment and RNA decay.

Dual-assignment of codons as termination and elongation codons is used to expand the genetic code. In mammals, UGA can be reassigned to selenocysteine during translation of selenoproteins by a mechanism involving a 3΄ untranslated region (UTR) selenocysteine insertion sequence (SECIS) and the SECIS-binding protein Secisbp2. Here, we present data from ribosome profiling, RNA-Seq and mRNA half-life measurements that support distinct roles for Secisbp2 in UGA-redefinition and mRNA stability. Conditional deletions of the Secisbp2 and Trsp (tRNASec) genes in mouse liver were compared to determine if the effects of Secisbp2 loss on selenoprotein synthesis could be attributed entirely to the inability to incorporate Sec. As expected, tRNASec depletion resulted in loss of ribosome density downstream of all UGA-Sec codons. In contrast, the absence of Secisbp2 resulted in variable effects on ribosome density downstream of UGA-Sec codons that demonstrate gene-specific differences in Sec incorporation. For several selenoproteins in which loss of Secisbp2 resulted in greatly diminished mRNA levels, translational activity and Sec incorporation efficiency were shown to be unaffected on the remaining RNA. Collectively, these results demonstrate that Secisbp2 is not strictly required for Sec incorporation and has a distinct role in stabilizing mRNAs that can be separated from its effects on UGA-redefinition.

Determining the fidelity of tRNA aminoacylation via microarrays.

The fidelity of tRNA aminoacylation is a critical determinant for the ultimate accuracy of protein synthesis. Although aminoacyl-tRNA synthetases are assumed to consistently maintain high tRNA charging fidelity, recent evidence has demonstrated that the fidelity of the aminoacylation reaction can be actively regulated and liable to change. Accordingly, the ability to conveniently assay the fidelity of tRNA charging is becoming increasingly relevant for studying mistranslation. Here we describe a combined radioactivity and microarray based method that can quantitatively elucidate which individual cognate or noncognate tRNA isoacceptors are charged with amino acid. In this technique, in vitro tRNA charging reactions or in vivo pulse-labeling is performed using a radiolabeled amino acid and tRNA microarrays are used to distinguish tRNA isoacceptors in total tRNA. During the tRNA array hybridization, each tRNA will hybridize to its unique probe and subsequent phosphorimaging of the array can determine which tRNAs were aminoacylated with the radiolabeled amino acid. The method can be used to assess the fidelity of tRNA charging in vivo or in vitro and can be applied to any organism with annotated tRNA genes.

Preterm neonatal urinary renal developmental and acute kidney injury metabolomic profiling: an exploratory study.

BACKGROUND: Acute kidney injury (AKI) staging has been developed in the adult and pediatric populations, but these do not yet exist for the neonatal population. Metabolomics was utilized to uncover biomarkers of normal and AKI-associated renal function in preterm infants. The study comprised 20 preterm infants with an AKI diagnosis who were matched by gestational age and gender to 20 infants without an AKI diagnosis. METHODS: Urine samples from pre-term newborn infants collected on day 2 of life were analyzed using broad-spectrum nuclear magnetic resonance (NMR) metabolomics. Multivariate analysis methods were used to identify metabolite profiles that differentiated AKI and no AKI, and to identify a metabolomics profile correlating with gestational age in infants with and without AKI. RESULTS: There was a clear distinction between the AKI and no-AKI profiles. Two previously identified biomarkers of AKI, hippurate and homovanillate, differentiated AKI from no-AKI profiles. Pathway analysis revealed similarities to cholinergic neurons, prenatal nicotine exposure on pancreatic ß cells, and amitraz-induced inhibition of insulin secretion. Additionally, a pH difference was noted. Both pH and the metabolites were found to be associated with AKI; however, only the metabotype was a significant predictor of AKI. Pathways for the no-AKI group that correlated uniquely with gestational age included aminoacyl-t-RNA biosynthesis, whereas pathways in the AKI group yielded potential metabolite changes in pyruvate metabolism. CONCLUSIONS: Metabolomics was able to differentiate the urinary profiles of neonates with and without an AKI diagnosis and metabolic developmental profiles correlated with gestational age. Further studies in larger cohorts are needed to validate these results.

Synthetic and editing reactions of aminoacyl-tRNA synthetases using cognate and non-cognate amino acid substrates.

The covalent coupling of cognate amino acid-tRNA pairs by corresponding aminoacyl-tRNA synthetases (aaRS) defines the genetic code and provides aminoacylated tRNAs for ribosomal protein synthesis. Besides the cognate substrate, some non-cognate amino acids may also compete for tRNA aminoacylation. However, their participation in protein synthesis is generally prevented by an aaRS proofreading activity located in the synthetic site and in a separate editing domain. These mechanisms, coupled with the ability of certain aaRSs to discriminate well against non-cognate amino acids in the synthetic reaction alone, define the accuracy of the aminoacylation reaction. aaRS quality control may also act as a gatekeeper for the standard genetic code and prevents infiltration by natural amino acids that are not normally coded for protein biosynthesis. This latter finding has reinforced interest in understanding the principles that govern discrimination against a range of potential non-cognate amino acids. This paper presents an overview of the kinetic assays that have been established for monitoring synthetic and editing reactions with cognate and non-cognate amino acid substrates. Taking into account the peculiarities of non-cognate reactions, the specific controls needed and the dedicated experimental designs are discussed in detail. Kinetic partitioning within the synthetic and editing sites controls the balance between editing and aminoacylation. We describe in detail steady-state and single-turnover approaches for the analysis of synthetic and editing reactions, which ultimately enable mechanisms of amino acid discrimination to be determined.