Breaking a single hydrogen bond in the mitochondrial tRNA -PheRS complex leads to phenotypic pleiotropy of human disease.

Various pathogenic variants in both mitochondrial tRNA and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNA . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element.

Kinetic and structural changes in HsmtPheRS, induced by pathogenic mutations in human FARS2.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS.

Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids.

Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on.

Universal pathway for posttransfer editing reactions: insights from the crystal structure of TtPheRS with puromycin.

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA(Phe) to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways.

The mechanistic and evolutionary aspects of the 2'- and 3'-OH paradigm in biosynthetic machinery.

Background: The translation machinery underlies a multitude of biological processes within the cell. The design and implementation of the modern translation apparatus on even the simplest course of action is extremely complex, and involves different RNA and protein factors. According to the "RNA world" idea, the critical link in the translation machinery may be assigned to an adaptor tRNA molecule.

Anticodon G recognition by tRNA synthetases mimics the tRNA core.

Ancient mechanisms for nucleotide base recognition in the RNA world are candidates for mimicking by early proteins like tRNA synthetases. In the core of the tRNA, conserved G22 interacts with two internal bases in a complex further stabilized by stacking interactions. This particular tRNA format for G recognition is shown here to be adapted by nine different and even nonhomologous anticodon binding domains (ABDs) of tRNA synthetases, in which amino acid side chains mimic all of the tRNA G22 base interactions.

Crystal structure of human mitochondrial PheRS complexed with tRNA(Phe) in the active "open" state.

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.

Bacterial and eukaryotic phenylalanyl-tRNA synthetases catalyze misaminoacylation of tRNA(Phe) with 3,4-dihydroxy-L-phenylalanine.

Aminoacyl-tRNA synthetases exert control over the accuracy of translation by selective pairing the correct amino acids with their cognate tRNAs, and proofreading the misacylated products. Here we show that three existing, structurally different phenylalanyl-tRNA synthetases-human mitochondrial (HsmtPheRS), human cytoplasmic (HsctPheRS), and eubacterial from Thermus thermophilus (TtPheRS), catalyze mischarging of tRNA(Phe) with an oxidized analog of tyrosine-L-dopa. The lowest level of L-dopa discrimination over the cognate amino acid, exhibited by HsmtPheRS, is comparable to that of tyrosyl-tRNA synthetase.

Common peptides study of aminoacyl-tRNA synthetases.

Background: Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains.

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP.

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution.

Structure of human cytosolic phenylalanyl-tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns.

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs.

Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS)-bacterial (αβ)(2), eukaryotic/archaeal cytosolic (αβ)(2), and mitochondrial α-is a prominent example of structural diversity within the aaRSs family.

'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains.

Large-scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl-tRNA synthetase.

Structural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a "closed" form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution.

Intra-protein compensatory mutations analysis highlights the tRNA recognition regions in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRSs) covalently attach amino acids to their corresponding nucleic acid adapter molecules, tRNAs. The interactions in the tRNA-aaRSs complexes are mostly non-specific, and largely electrostatic. Tracing a way of aaRS-tRNA mutual adaptation throughout evolution offers a clearer view of understanding how aaRS-tRNA systems preserve patterns of tRNA recognition and binding.

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta-tyrosine.

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins.

Crystallization and X-ray analysis of human cytoplasmic phenylalanyl-tRNA synthetase.

Human cytosolic phenylalanyl-tRNA synthetase (hcPheRS) is responsible for the covalent attachment of phenylalanine to its cognate tRNA(Phe). Significant differences between the amino-acid sequences of eukaryotic and prokaryotic PheRSs indicate that the domain composition of hcPheRS differs from that of the Thermus thermophilus analogue. As a consequence of the absence of the anticodon-recognizing B8 domain, the binding mode of tRNA(Phe) to hcPheRS is expected to differ from that in prokaryotes.

The tRNA-induced conformational activation of human mitochondrial phenylalanyl-tRNA synthetase.

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea.

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids.

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis.

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase.

Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial alpha-subunit of PheRS and the B8 domain of its beta-subunit. Together, the alpha-subunit and the 'RNP-domain' (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity.

The crystal structure of the ternary complex of phenylalanyl-tRNA synthetase with tRNAPhe and a phenylalanyl-adenylate analogue reveals a conformational switch of the CCA end.

The crystal structure of the ternary complex of (alphabeta)(2) heterotetrameric phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus with cognate tRNA(Phe) and a nonhydrolyzable phenylalanyl-adenylate analogue (PheOH-AMP) has been determined at 3.1 A resolution. It reveals conformational changes in tRNA(Phe) induced by the PheOH-AMP binding.

Optimal growth temperature of prokaryotes correlates with class II amino acid composition.

Partitioning of aminoacyl-tRNA synthetases and their associated amino acids into two classes allows us to distinguish between thermophilic and mesophilic species based only on amino acids composition. The CLASSDB program has been developed for amino acid content analysis in organisms treated individually or pooled together to form a pattern of characteristic properties. A strong correlation has been observed between optimal growth temperature (OGT) of organisms and class II amino acids content.

Structural basis for discrimination of L-phenylalanine from L-tyrosine by phenylalanyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) exert control over the faithful transfer of amino acids onto cognate tRNAs. Since chemical structures of various amino acids closely resemble each other, it is difficult to discriminate between them. Editing activity has been evolved by certain aaRSs to resolve the problem.

Electrostatic potential of aminoacyl-tRNA synthetase navigates tRNA on its pathway to the binding site.

In the first stage of a diffusion-controlled enzymatic reaction, aminoacyl-tRNA synthetases (aaRSs) interact with cognate tRNAs forming non-specific encounters. The aaRSs catalyzing the same overall aminoacylation reaction vary greatly in subunit organization, structural domain composition and amino acid sequence. The diffusional association of aaRS and tRNA was found to be governed by long-range electrostatic interactions when the homogeneous negative potential of tRNA fits to the patches of positive potential produced by aaRS; one patch for each tRNA substrate molecule.