Aminoacyl Transfer Research Articles

Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases.

Cyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs.

Toward peptide nucleic acid (PNA) directed peptide translation using ester based aminoacyl transfer.

Peptide synthesis is a fundamental feature of life. However, it still remains unclear how the contemporary translation apparatus evolved from primitive prebiotic systems and at which stage of the evolution peptide synthesis emerged. Using simple molecular architectures, in which aminoacyl transfer of phenylalanine occurs either between two ends of a PNA stem loop structure, between two PNAs in a duplex, or between two PNAs assembled on a PNA template, we show that bona fide template instructed phenylalanine transfer can take place. Thus, we have identified conditions which allow template assisted intermolecular aminoacyl transfer using simple ester aminolysis chemistry primitively analogous to the ribosomal peptidyl transferase reaction in the absence of anchimeric assistance from ribose and ribosome catalysis. These results help define the minimum chemical boundary conditions for the translation process and also give insight into the possibilities for the prebiotic emergence of RNA-independent translation.

Evolving from an Enzyme and into a Regulator

Alternative splicing of aminoacyl transfer RNA synthetases ablates the catalytic domain to yield diverse alternative functions.

Nucleic acid catalysis: metals, nucleobases, and other cofactors.

The discovery of RNA-catalyzed phosphodiester bond cleavage by Cech and Altman in 1982–1983 shattered the paradigm of protein-dependent biological catalysis and opened up new horizons for RNA biology (reviewed in ref (1)). These discoveries ushered in a new era of high activity in RNA biochemistry and structural biology that, in conjunction with developments in genomics, established groundwork for the ensuing explosion in discoveries of other functional but noncoding RNAs. Since the initial discoveries, several classes of naturally occurring and artificially developed ribozymes have been defined. Ribozyme reactions catalyzed in nature include phosphoryl and aminoacyl transfer reactions (Figure ​(Figure1).1). RNA-catalyzed reactions obtained in vitro extend to carbon–carbon bond formation in the Diels–Alderase ribozyme. In vitro selection has also been employed to discover DNA-based enzymes that catalyze a number of different reactions, including RNA cleavage, as in Figure ​Figure11A. Figure 1 Reactions catalyzed by naturally occurring ribozymes. (A) Intramolecular phosphoryl-transfer reaction catalyzed by the “nucleolytic” ribozymes including the hammerhead, hepatitis delta virus (HDV), hairpin, Neurospora VS, and GlmS RNAs. ...

Establishment of an ELISA to detect anti-glycyl-tRNA synthetase antibody (anti-EJ), a serological marker of dermatomyositis/polymyositis and interstitial lung disease

BackgroundThe aminoacyl transfer RNA synthetases (ARSs) are a group of enzymes that charge amino acids to the cognate transfer RNA during the translation process. Previous reports demonstrated autoantibodies to 8 different ARS. Although anti-glycyl-tRNA synthetase antibodies (anti-EJ) are mainly found in patients with inflammatory myopathy, information on their clinical significances is limited, partly due to a lack of commercially available tests. MethodsWe developed an ELISA and immunoprecipitation method by using recombinant EJ protein to detect the anti-EJ of 453 patients with various autoimmune connective tissue diseases (ACTDs). We also studied the influence of 3 cytokines—IL-1β, IFN-γ and IFN-α—on the level of EJ mRNA and protein expressed by human fetal lung fibroblasts. ResultsFive patients were positive for anti-EJ. Although 3 of these patients had dermatomyositis/polymyositis, the other 2 patients did not have myositis. The three patients with high levels of anti-EJ antibodies in ELISA were complicated with interstitial lung disease. There was no significant change in the level of EJ protein expressed by human fetal lung fibroblasts stimulated by the cytokines. ConclusionWe developed an ELISA to detect anti-EJ by using recombinant protein. This easy-to-use ELISA could help clarify the clinical significance of anti-EJ in ACTDs.

Clinical Phenotype of Japanese Patients with Dermatomyositis—Classification Based on Dermatomyositis-Specific Autoantibodies

Objectives: To correlate the precise specificity of autoantibodies in Japanese dermatomyositis (DM) patients with their clinical phenotypes. Methods: Serum samples from 94 adult DM patients (67 with classical DM and 27 with clinically amyopathic dermatomyositis, CADM) were screened for autoantibodies using immunoprecipitation assays. Patients with antibodies against aminoacyl transfer RNA synthetase (ARS), Mi-2 or who had other autoantibodies were assessed for clinical symptoms and laboratory findings. Results: Sera from 27 of 94 DM patients (29%) were found to have anti-ARS antibodies. Nineteen (20%) had anti-CADM-140/MDA5, 5 (5%) had anti-Mi-2, and 8 (6%) had anti-p155/TIF1-γ. Anti-MJ/NXP-2 was not found in our series of adult DM. Seventeen patients with anti-ARS had fever and 22 had arthritis and interstitial lung disease (ILD), compatible with a diagnosis of anti-ARS syndrome. Seventeen of 19 (89%) with anti-CADM-140/MDA5 had ILD, 16 (84%) of whom developed rapidly progressive ILD (RP-ILD). Four of 5 (80%) with anti-Mi-2 had heliotrope rash and/or Gottron’s sign/papules, and 2 (40%) had V-sign and/or shawl-sign rash, whereas no ILD or malignancy was detected. As seen with anti-Mi-2-positive patients, a low frequency of ILD (13%) was found in patients with anti-p155/TIF1-γ but 6 of 8 (75%) had malignancy during their course. The frequency of ILD was significantly higher in patients with anti-ARS or anti-CADM-140/MDA5 compared with anti-Mi-2 or anti-p155/TIF1-γ (81% and 89%, respectively). It should be noted that anti-CADM-140/MDA5-positive patients suffered significantly more RP-ILD compared to patients with anti-ARS (84% vs. 7%, P < 0.0001). On the other hand, anti-p155/TIF1-γ positive patients had a significantly higher rate of malignancy compared with anti-ARS-, anti-CADM-140/MDA5-and anti-Mi-2-positive patients (75% vs. 7%: P = 0.0004, 5%: P = 0.0006, 0%: P = 0.02, respectively). Conclusions: These results indicate that in addition to antibodies previously identified as specific for DM, autoantibodies newly found in these patients are useful for stratifying them into clinical subgroups.

The Structure of FemXWvin Complex with a Peptidyl-RNA Conjugate: Mechanism of Aminoacyl Transfer from Ala-tRNAAlato Peptidoglycan Precursors

The Structure of FemX_Wvin Complex with a Peptidyl-RNA Conjugate: Mechanism of Aminoacyl Transfer from Ala-tRNA^Alato Peptidoglycan Precursors

The Structure of FemXWv in Complex with a Peptidyl‐RNA Conjugate: Mechanism of Aminoacyl Transfer from Ala‐tRNAAla to Peptidoglycan Precursors

The Structure of FemX_Wv in Complex with a Peptidyl‐RNA Conjugate: Mechanism of Aminoacyl Transfer from Ala‐tRNA^Ala to Peptidoglycan Precursors

Autoantibodies to transcription intermediary factor 1 in dermatomyositis shed insight into the cancer–myositis connection

Across the breadth of autoimmune rheumatic diseases, there is a striking association of specific autoantibodies with distinct clinical phenotypes, making them excellent tools for classifying patients and predicting disease course and outcomes. Within the myositis spectrum, there are numerous examples of this. More than 2 decades ago, it was noted that autoantibodies against the aminoacyl–transfer RNA synthetases (the most frequently targeted of which is Jo-1) are found in myositis patients with a constellation of symptoms known as the “synthetase syndrome” (1,2). These include mechanic’s hands, interstitial lung disease, inflammatory arthritis, Raynaud’s phenomenon, and fever. A more recently described specificity is that of melanoma differentiation–associated protein 5 (MDA-5). Antibodies to MDA-5 are found in dermatomyositis (DM) patients with mild/absent muscle disease and are frequently associated with rapidly progressive interstitial lung disease (3). New data continue to further define the clinical phenotype associated with anti–MDA-5 antibodies; in a study of DM patients seen at an outpatient dermatology clinic, our group demonstrated that this specificity is associated with cutaneous ulcerations and distinctive palmar papules, and confirmed the association with rapidly progressing lung disease (4). These 2 examples confirm that autoantibodies in DM patients are of clinical utility. Autoantibodies against a protein doublet termed “p155/140” (denoting the molecular weights) define another distinct group of DM patients—those with an increased incidence of cancer compared to DM patients without malignancies (5). A meta-analysis of several studies confirmed that the presence of these autoantibodies has 70% sensitivity and 89% specificity for identifying patients with cancer-associated DM (6). This immune response appears specific to DM, as it is not found in patients with systemic sclerosis, lupus erythematosus, or healthy individuals. In 2006, p155 was identified as transcription intermediary factor 1 (TIF-1 ) by Targoff et al (7), but the identity of the 140-kd specificity remained elusive. The current study by Fujimoto et al, reported in this issue of Arthritis & Rheumatism (8), confirms the identity of p155 as TIF-1 (consistent with Targoff and colleagues’ findings described above) and also identifies the 140-kd antibody target as TIF-1 . The study also shows that TIF-1 (100 kd) is targeted in DM patients, although less frequently than its TIF-1 and TIF-1 counterparts. The TIF-1 specificities occurred alone (in the case of TIF-1 and TIF-1 , but not TIF-1 ) or in various combinations of TIF-1 , TIF-1 , and TIF-1 ; of the 78 DM patients studied by Fujimoto et al, 62% had anti–TIF-1 and anti–TIF-1 , 29% had anti– TIF-1 alone, 5.1% had antibodies to all 3 proteins, 2.6% had anti–TIF-1 and anti–TIF-1 , and 1.3% had anti–TIF-1 alone. Since these proteins are highly homologous, and because anti–TIF-1 frequently occurs alone while anti–TIF-1 never does, one possibility is that the epitope is a TIF-1 sequence, with crossreactivity of the antibodies to TIF-1 . Interesting in this regard is the fact that TIF-1 and TIF-1 share much more sequence similarity than with TIF-1 , perhaps explaining why anti–TIF-1 antibodies are so infrequently detected. These DM-specific antibodies are relatively frequent, being found in 17% of DM patients (78 of 456) in the study by Fujimoto et al. They constitute significant subsets in juvenile DM and adult cancer-associated DM Supported by the NIH (grant R01-AR-44684 to Dr. CasciolaRosen). David Fiorentino, MD, PhD: Stanford University School of Medicine, Stanford, California; Livia Casciola-Rosen, PhD: Johns Hopkins University School of Medicine, Baltimore, Maryland. Address correspondence to Livia Casciola-Rosen, PhD, Johns Hopkins University School of Medicine, Division of Rheumatology, 5200 Eastern Avenue, Mason F. Lord Building, Center Tower Suite 5300, Baltimore, MD 21224. E-mail: lcr@jhmi.edu. Submitted for publication September 22, 2011; accepted in revised form October 4, 2011.

Small aminoacyl transfer centers at GU within a larger RNA

Separate aminoacyl transfer centers related to the small …GUNNN..: NNNU ribozyme seem possible at the frequent GU sequences dispersed throughout an RNA tertiary structure. In fact, such activity is easily detected and varies more than 2 orders in rate, probabably being faster at sites with less structural constraint. Analysis of a particular constrained active site in an rRNA transcript suggests that its difficulty lies not in substrate strand association, but in binding and/or group transfer from the aminoacyl precursor. Efficient aminoacyl transfer requires accurate complementarity between large or small ribozymes and oligoribonucleotide substrates, even when only three or four base pairs link the two. Thus, multi-site active ribozymal superstructures might have coordinated an RNA metabolism, including aiding an early translation apparatus.

Unique domain appended to vertebrate tRNA synthetase is essential for vascular development

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT.

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.

Response to Comment on “The Mechanism for Activation of GTP Hydrolysis on the Ribosome”

Our report of the crystal structure of elongation factor Tu (EF-Tu) and aminoacyl–transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog included a proposed mechanism of GTP hydrolysis by EF-Tu involving histidine-84. Liljas et al . summarize experimental evidence against this mechanism and propose a substrate-assisted catalytic model. However, these experiments and the model are also problematic. Further study is required to definitively determine the mechanism of GTP hydrolysis by EF-Tu.

Comment on “The Mechanism for Activation of GTP Hydrolysis on the Ribosome”

Voorhees et al. (Reports, 5 November 2010, p. 835) determined the structure of elongation factor Tu (EF-Tu) and aminoacyl-transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog. However, their identification of histidine-84 of EF-Tu as deprotonating the catalytic water molecule is problematic in relation to their atomic structure; the terminal phosphate of GTP is more likely to be the proper proton acceptor.

An idiosyncratic serine ordering loop in methanogen seryl-tRNA synthetases guides substrates through seryl-tRNASer formation

Seryl-tRNA synthetases (SerRS) covalently attach serine to cognate tRNASer. Atypical SerRSs, considerably different from canonical enzymes, have been found in methanogenic archaea. A crystal structure of methanogenic-type SerRS revealed a motif within the active site (serine ordering loop; SOL), which undergoes a notable induced-fit rearrangement during serine binding. The loop rearranges from a disordered conformation in the unliganded enzyme, to an ordered structure comprising an α-helix followed by a loop. We performed kinetic and thermodynamic analyses of SerRS variants to establish the role of the SOL in serylation. Thermodynamic data confirmed a linkage between binding of serine and α-helix formation, previously described by the crystallographic analysis. The ability of the SOL to adopt the observed secondary structure was recognized as essential for serine activation. Mutation of Gln400, which according to the structural data establishes the main connection between the serine and the SOL, produced only modest kinetic effects. Kinetic data offer new insights into the coupling of the conformational change with active site assembly. Productive positioning of the SOL may be driven by the interaction between Trp396 and the serine α-amino group. Rapid kinetics reveals that His250, a non-SOL residue, is essential for transfer of serine to tRNA. Modeling data established that accommodation of the tRNA within the active site may require movement of the SOL. This would enable His250 to assist in productive positioning of the 3′-end of the tRNA for the aminoacyl transfer. Thus, the rearrangements of the SOL conformationally adjust the active site for both reaction steps.

Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some nonproteogenic amino acids, including β-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by Escherichia coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k(cat)/K(M)) for HNV was found to be only 20-30-fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s(-1)) was 10-fold higher than the multiple turnover k(cat) value (1 s(-1)), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like noncognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by nonaminoacylatable (3'-dA76) tRNA. ThrRS also failed to exhibit posttransfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against nonproteogenic amino acids that are not encountered under normal physiological conditions. Other examples of "fidelity escape" in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.

Intrinsic pKa values of 3′-N-α-l-aminoacyl-3′-aminodeoxyadenosines determined by pH dependent 1H NMR in H2O

3'-(α-L-Aminoacylamido)deoxyadenosines are ribosomal A-site binders and mimic the nascent peptide accepting 3'-terminus of aminoacyl transfer RNA. Their α-amino groups exhibit intrinsic basicities in bulk water that differ by up to 1.8 pK(a) units. Only the neutral form of these nucleophiles can be active during ribosomal peptidyl transfer catalysis.

A Likely Conformation

At several stages of protein synthesis, guanosine triphosphate hydrolyzing enzymes (GTPases) interact with the ribosome, and GTP hydrolysis is coupled to progression of synthesis. Voorhees et al. (p. [835][1]) have determined a 3.2-resolution structure of the GTPase, elongation factor Tu, which delivers amino-acyl transfer RNA (tRNA) to the ribosome. The GTPase and tRNA were bound to the ribosome and were stalled in an active conformation by a GTP analog. The structure revealed that activation of the enzyme only required small changes in conformation to move a catalytic histidine into the correct position for hydrolysis. A similar mechanism likely applies to the activation of other translational GTPases. [1]: /lookup/doi/10.1126/science.1194460

The Mechanism for Activation of GTP Hydrolysis on the Ribosome

Protein synthesis requires several guanosine triphosphatase (GTPase) factors, including elongation factor Tu (EF-Tu), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. To understand how the ribosome triggers GTP hydrolysis in translational GTPases, we have determined the crystal structure of EF-Tu and aminoacyl-tRNA bound to the ribosome with a GTP analog, to 3.2 angstrom resolution. EF-Tu is in its active conformation, the switch I loop is ordered, and the catalytic histidine is coordinating the nucleophilic water in position for inline attack on the γ-phosphate of GTP. This activated conformation is due to a critical and conserved interaction of the histidine with A2662 of the sarcin-ricin loop of the 23S ribosomal RNA. The structure suggests a universal mechanism for GTPase activation and hydrolysis in translational GTPases on the ribosome.

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA(AA) formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.