α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain.

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.

Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation.

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

Direct Measurements of Erythromycin's Effect on Protein Synthesis Kinetics in Living Bacterial Cells.

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.

A novel, mitochondrial, internal tRNA-derived RNA fragment possesses clinical utility as a molecular prognostic biomarker in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults. The prognosis of CLL patients varies considerably. Transfer RNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNA fragments excised from mature tRNAs and pre-tRNAs located in nuclei as well as in mitochondria. In this study, the clinical utility of i-tRF-PheGAA, a novel mitochondrial tRF, was investigated in CLL. DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CLL patients and 43 non-leukemic controls. Total RNA was isolated from each sample, polyadenylated at the 3' end and reversely transcribed. An in-house developed real-time quantitative PCR assay was developed and applied, and the results were biostatistically analyzed. For the normalization of the i-tRF-PheGAA expression levels, the expression of a small nucleolar RNA (RNU48) was used as reference. RESULTS: Mann-Whitney U test showed that i-tRF-PheGAA can distinguish between CLL samples and normal controls (p < 0.001). As determined by Kaplan-Meier survival analysis, overexpression of i-tRF-PheGAA was related to poor overall survival of the CLL patients (p < 0.001). Univariate bootstrap Cox regression analysis exhibited a higher hazard ratio of 7.95 (95% CI = 2.37-26.72, p < 0.001) for patients with positive i-tRF-PheGAA expression status. Multivariate bootstrap Cox regression analysis showed that the prognostic value of this tRF is independent of clinical stage, mutational status of the immunoglobulin heavy chain variable (IGHV) genetic locus, and CD38 expression status (p = 0.010). CONCLUSIONS: Our results show that i-tRF-PheGAA can serve as a molecular biomarker of poor prognosis in CLL, alongside with the existing factors for CLL prognosis.

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress.

BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.

Structural and thermodynamic analysis of the binding of tRNA(phe) by the putative anticancer alkaloid chelerythrine: Spectroscopy, calorimetry and molecular docking studies.

The interaction of the putative anticancer alkaloid chelerythrine with tRNA(phe) was characterized by spectroscopy, calorimetry and molecular docking studies. The charged iminium form of chelerythrine binds with tRNA(phe) in a cooperative mode with a binding affinity value of (4.06±0.01)×10(5)M(-1). The neutral alkanolamine form does not bind to tRNA(phe) but in the presence of high concentration of tRNA(phe) this form gets converted to the iminium form and then binds with tRNA(phe). The partial intercalative mode of binding of chelerythrine to the tRNA(phe) was characterized from the steady state anisotropy, iodide ion-induced fluorescence quenching and viscosity measurements. Chelerythrine binding induced conformational perturbations in tRNA(phe) as observed from the circular dichroism spectroscopy. The strong binding was also supported by the ethidium bromide displacement assay. The binding was favoured by both enthalpy and entropy contributions. Although the binding was dependent on the [Na(+)], non-electrostatic forces contributed predominantly to the Gibbs energy change. The negative value of the heat capacity change proposed the involvement of hydrophobic forces in the binding. Molecular docking study was carried out to decipher the details of the recognition of tRNA(phe) by chelerythrine. The study provided insights about the chelerythrine binding pockets on tRNA(phe) and marked the necessary interactions for binding of chelerythrine molecule. Partially intercalative mode of the alkaloid binding was supported by docking studies. In total, docking studies corroborated well with our experiential observations. The structural and thermodynamic results of chelerythrine binding to tRNA(phe) may be helpful to develop new RNA therapeutic agents.

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase.

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

Mechanistic Studies of the Radical S-Adenosyl-L-methionine Enzyme 4-Demethylwyosine Synthase Reveal the Site of Hydrogen Atom Abstraction.

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m¹G) with carbons 2 and 3 of pyruvate. In this study, labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a ²H-labeled m¹G methyl group was used to identify the methyl group of m¹G as the site of hydrogen atom abstraction by 5'-deoxyadenosyl radical. [2-¹³C1-3,3,3-²H3]Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.

Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation.

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNA(i)(met) and elongator tRNA(phe) are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNA(i)(met) in the absence of ribosomal bases. The misacylated elongator tRNA(phe), however, did not show any difference in its PES compared with that for the phe-tRNA(phe). This demonstrates the structural specification of initiator tRNA(i)(met) for the amino acids side chain.

Structure-function analysis of human TYW2 enzyme required for the biosynthesis of a highly modified Wybutosine (yW) base in phenylalanine-tRNA.

Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNA(Phe). TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNA(Phe). Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNA(Phe) modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.

Binding of the anticancer alkaloid sanguinarine with tRNA(phe): spectroscopic and calorimetric studies.

The interaction of the natural plant alkaloid and anticancer agent sanguinarine with tRNA(phe) has been investigated by spectroscopic and calorimetric techniques. Sanguinarine iminium binds to tRNA(phe) cooperatively; alkanolamine does not bind but in presence of large tRNA(phe) concentration, a conversion from alkanolamine to iminium occurs resulting in concomitant binding of the latter. The binding affinity of the iminium to tRNA(phe) obtained from isothermal titration calorimetry was of the order of 10(5) M(-1), which is close to that evaluated from spectroscopy. The binding was driven largely by negative enthalpy and a smaller but favourable positive entropy change. The binding was dependent on the [Na(+)] concentration, but had a larger non-electrostatic contribution to the Gibbs energy. A small heat capacity value and the enthalpy-entropy compensation in the energetics of the interaction characterized the binding of the iminium form to tRNA(phe). This study confirms that the tRNA(phe) binding moiety is the iminium form of sanguinarine.

Targeting RNA by small molecules: comparative structural and thermodynamic aspects of aristololactam-ß-D-glucoside and daunomycin binding to tRNA(phe).

BACKGROUND: Interaction of aristololactam-ß-D-glucoside and daunomycin with tRNA(phe) was investigated using various biophysical techniques. METHODOLOGY/PRINCIPAL FINDINGS: Absorption and fluorescence studies revealed that both the compounds bind tRNA(phe) non-cooperatively. The binding of daunomycin was about one order of magnitude higher than that of aristololactam-ß-D-glucoside. Stronger binding of the former was also inferred from fluorescence quenching data, quantum efficiency values and circular dichroic results. Results from isothermal titration calorimetry experiments suggested that the binding of both compounds was predominantly entropy driven with a smaller but favorable enthalpy term that increased with temperature. A large favorable electrostatic contribution to the binding of daunomycin to tRNA(phe) was revealed from salt dependence data and the dissection of the free energy values. The electrostatic component to the free energy change for aristololactam-ß-D-glucoside-tRNA(phe) interaction was smaller than that of daunomycin. This was also inferred from the slope of log K versus [Na(+)] plots. Both compounds enhanced the thermal stability of tRNA(phe). The small heat capacity changes of -47 and -99 cal/mol K, respectively, observed for aristololactam-ß-D-glucoside and daunomycin, and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak noncovalent interactions. Molecular aspects of the interaction have been revealed. CONCLUSIONS/SIGNIFICANCE: This study presents the structural and energetic aspects of the binding of aristololactam-ß-D-glucoside and daunomycin to tRNA(phe).

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.

Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase.

Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth's history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded ß-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily.

Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2.

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an alpha-amino-alpha-carboxypropyl ("acp") group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the "acp" group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNA(Phe), and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this "acp" group has a significant role in preventing decoding frame shifts, the mechanism of the "acp" group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the "acp" group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the "acp" group, and not the methyl group, from AdoMet to the nucleobase.

Structural basis of tRNA modification with CO2 fixation and methylation by wybutosine synthesizing enzyme TYW4.

Wybutosine (yW), one of the most complicated modified nucleosides, is found in the anticodon loop of eukaryotic phenylalanine tRNA. This hypermodified nucleoside ensures correct codon recognition by stabilizing codon-anticodon pairings during the decoding process in the ribosome. TYW4 is an S-adenosylmethionine (SAM)-dependent enzyme that catalyzes the final step of yW biosynthesis, methylation and methoxycarbonylation. However, the structural basis for the catalytic mechanism by TYW4, and especially that for the methoxycarbonylation, have remained elusive. Here we report the apo and cofactor-bound crystal structures of yeast TYW4. The structures revealed that the C-terminal domain folds into a beta-propeller structure, forming part of the binding pocket for the target nucleoside. A comparison of the apo, SAM-bound, and S-adenosylhomocysteine-bound structures of TYW4 revealed a drastic structural change upon cofactor binding, which may sequester solvent from the catalytic site during the reaction and facilitate product release after the reaction. In conjunction with the functional analysis, our results suggest that TYW4 catalyzes both methylation and methoxycarbonylation at a single catalytic site, and in the latter reaction, the methoxycarbonyl group is formed through the fixation of carbon dioxide.

Antisense oligonucleotides targeting universally conserved 26S rRNA domains of plant ribosomes at different steps of polypeptide elongation.

A ribosome undergoes significant conformational changes during elongation of polypeptide chain that are correlated with structural changes of rRNAs. We tested nine different antisense oligodeoxynucleotides complementary to the selected, highly conserved sequences of Lupinus luteus 26S rRNA that are engaged in the interactions with tRNA molecules. The ribosomes were converted either to pre- or to posttranslocational states, with or without prehybridized oligonucleotides, using tRNA or mini-tRNA molecules. The activity of those ribosomes was tested via the so-called binding assay. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis. In this article, we present that (i) before and after translocation, fragments of domain V between helices H70/H71 and H74/H89 do not have to interact with nucleotides 72-76 of the acceptor arm of A-site tRNA; (ii) helix H69 does not have to interact with DHU arm of tRNA in positions 25 and 26 after forming the peptide bond, but before translocation; (iii) helices H69 and H70 interact weakly with nucleotides 11, 12, 25, and 26 of A-site tRNA before forming a peptide bond in the ribosome; (iv) interactions between helices H80, H93 and single-stranded region between helices H92 and H93 and CCAend of P-site tRNA are necessary at all steps of elongation cycle; and (v) before and after translocation, helix H89 does not have to interact with nucleotides in positions 64-65 and 50-53 of A-site tRNA TPsiC arm.