Transfer RNA Recognition by Class I Lysyl-tRNA Synthetase from the Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi and other spirochetes contain a class I lysyl‐tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNALys recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNALys in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNALys were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNAAsp scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined

Plasmodium apicoplast tyrosyl-tRNA synthetase recognizes an unusual, simplified identity set in cognate tRNATyr

The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity.publishe

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.comparative studyjournal articleresearch support, non-u.s. gov't1994 Jul 25importe

Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase.

Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA(Tyr) charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4(3)2(1)2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 A resolution. Complete data sets could be collected and led to structure solution by molecular replacement.journal articleresearch support, non-u.s. gov't2007 Apr 012007 03 30importe

Exploration au coeur de la machinerie traductionnelle de Plasmodium falciparum (Etude de domaines de liaison aux ARN de transfert)

Ce travail de thèse concerne deux protéines impliquées dans la traduction chez Plasmodium falciparum, parasite responsable du paludisme chez l Homme. Elles ont en commun de reconnaître les ARN de transfert (ARNt): (i) l aspartyl-ARNt synthètase (AspRS) reconnaît spécifiquement l ARNtAsp et catalyse l attachement de l acide aspartique sur son extrémité 3 et (ii) une protéine de fonction inconnue, appelée PftRBP (tRNA binding protein) qui elle, interagit avec tous les ARNt.L AspRS cytoplasmique de P. falciparum, qui s accumule au cours du stade sanguin du développement du parasite est plus courte que ne l indique la banque de données génomique. De plus, deux domaines fonctionnels caractéristiques de cette enzyme ont été identifiés : un motif riche en lysines dans l extension N-terminale et une courte insertion présente dans le domaine de liaison à l anticodon. Comme ces deux domaines, essentiels pour l activité de l AspRS du parasite, sont absents dans son homologue humain, ils ont été utilisés dans diverses approches prometteuses pour la recherche de molécules pourvues de propriétés potentiellement antipaludiques.La deuxième protéine, PftRBP n est retrouvé que chez Plasmodium et comporte un domaine de liaison aux ARNt à son extrémité C-terminale. Nous avons pu montrer in vitro que PftRBP: (i) lie spécifiquement et avec une forte affinité tous les ARNt, (ii) reconnaît le coude formé par les boucles D et T dans la structure conservée en L des ARNt, (iii) s organise en tétramère et (iv) est localisée à la surface du parasite. L ensemble de ces données suggère une fonction unique et originale pour cette protéine impliquant un trafic d ARNt entre le parasite et son hôte.This PhD work concentrates on two proteins of the human malaria parasite Plasmodium falciparum. They are both involved in the protein synthesis process of this parasite and interact with transfer RNAs (tRNAs): (i) aspartyl-tRNA synthetase (AspRS) specifically recognizes tRNAAsp and catalyzes the binding of aspartic acid to its 3 end and (ii) a protein of unknown function, PftRBP (tRNA binding protein), that interacts with all tRNAs.It has been shown that the plasmodial AspRS, which accumulates during the erythrocytic stage of the parasite is shorter than expected, based on the genome database. This AspRS has two unique functional domains: a lysine-rich tRNA binding motif in the N-terminal extension of the protein and a short insertion in the anticodon binding domain. It has been demonstrated that these two motifs are essential for the parasite s AspRS activity. Since they are absent in the human homologue, they were targeted to identify specific inhibitors with putative antimalarial effects.The second protein, PftRBP, displays a tRNA binding domain at its C-terminus that binds to all tRNA sequences. Indeed, in vitro PftRBP (i) binds only tRNAs with a high affinity, (ii) recognizes the elbow formed by the D and T loops of the conserved tRNA L-shaped structure, (iii) forms a tetramer and (iv) is exposed at the parasite s surface. These results suggest a unique and original function for this protein implicating tRNA trafficking between the parasite and its host.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Human mitochondrial TyrRS disobeys the tyrosine identity rules

Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNA(Tyr) and the reverse pair C1-G72 present in archaeal and eukaryal tRNA(Tyr). This expectation was verified by a functional analysis of wild-type or mutated tRNA(Tyr) molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNA(Tyr) with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs

Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants

In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed “pseudo-WT“ tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson–Crick pairs into G–U pairs or C•A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders

Atypical archaeal tRNA pyrrolysine transcript behaves towards EF-Tu as a typical elongator tRNA

The newly discovered tRNA(Pyl) is involved in specific incorporation of pyrrolysine in the active site of methylamine methyltransferases in the archaeon Methanosarcina barkeri. In solution probing experiments, a transcript derived from tRNA(Pyl) displays a secondary fold slightly different from the canonical cloverleaf and interestingly similar to that of bovine mitochondrial tRNA(Ser)(uga). Aminoacylation of tRNA(Pyl) transcript by a typical class II synthetase, LysRS from yeast, was possible when its amber anticodon CUA was mutated into a lysine UUU anticodon. Hydrolysis protection assays show that lysylated tRNA(Pyl) can be recognized by bacterial elongation factor. This indicates that no antideterminant sequence is present in the body of the tRNA(Pyl) transcript to prevent it from interacting with EF-Tu, in contrast with the otherwise functionally similar tRNA(Sec) that mediates selenocysteine incorporation

Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetases.

High-resolution X-ray structures for the tRNA/aminoacyl-tRNA synthetase complexes between Escherichia coli tRNAGln/GlnRS and yeast tRNAAsp/AspRS have been determined. Positive identity nucleotides that direct aminoacylation specificity have been defined in both cases; E. coli tRNAGln identity is governed by 10 elements scattered in the tRNA structure, while specific aminoacylation of yeast tRNAAsp is dependent on 5 positions. Both identity sets are partially overlapping and share 3 nucleotides. Interestingly, the two enzymes belong to two different classes described for aminoacyl-tRNA synthetases. The class I glutaminyl-tRNA synthetase and the class II aspartyl-tRNA synthetase recognize their cognate tRNA from opposite sides. Mutants derived from glutamine and aspartate tRNAs have been created by progressively introducing identity elements from one tRNA into the other one. Glutaminylation and aspartylation assays of the transplanted tRNAs show that identity nucleotides from a tRNA originally aminoacylated by a synthetase from one class are still recognized if they are presented to the enzyme in a structural framework corresponding to a tRNA aminoacylated by a synthetase belonging to the other class. The simple transplantation of the glutamine identity set into tRNAAsp is sufficient to obtain glutaminylatable tRNA, but additional subtle features seem to be important for the complete conversion of tRNAGln in an aspartylatable substrate. This study defines C38 in yeast tRNAAsp as a new identity nucleotide for aspartylation. We show also in this paper that, during the complex formation, aminoacyl-tRNA synthetases are at least partially responsible for conformational changes which involve structural constraints in tRNA molecules.journal articleresearch support, non-u.s. gov'tresearch support, u.s. gov't, p.h.s.1994 Aug 23importe