NMR studies of telomeric nucleoprotein complexes involving the Myb-like domain of the human telomeric protein TRF2

In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1

The Human Mitochondrial tRNAMet: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNAMetCAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl-(A-) sites of the ribosome. The hmtRNAMetCAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f5C34), and a cytidine substituting for the invariant uridine at position 33 of the canonical “U-turn” in tRNAs. The structure of the tRNA's anticodon stem and loop domain (hmtASLMetCAU), determined by NMR restrained molecular modeling, revealed how the f5C34 modification facilitates the decoding of AUA at the P- and A-sites. The f5C34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASLMetCAU. The hmtASLMetCAU exhibited a “C-turn” conformation that has some characteristics of the U-turn motif. Codon binding studies with both E. coli and bovine mitochondrial ribosomes revealed that the f5C34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL's binding kinetics. Mitochondrial translation by many organisms including humans sometimes initiates with the universal isoleucine codons AUU and AUC. The f5C34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f5C34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria

The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNALys3, to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3′-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5′-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNALys3. The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s2U34, and pseudouridine, Ψ39, appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U162•Ψ39 and G163•A38, that maintained a reasonable A-form helix diameter. The tRNA's s2U34 stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Ψ39 stabilized the adjacent mismatched pairs

tRNA structural and functional changes induced by oxidative stress

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level

Etude de complexes télomériques par Résonance Magnétique Nucléaire

Telomeres are special nucleoprotein complexes at the end of eukaryotic chromosomes. Human telomeric DNAcontains between 500 and 3000 repeats of the sequence d(T2AG3)n (G-strand) and of the complementary strand C3TA2 (C-strand). The G-strand forms at the end of telomeres a single strand overhang containing 50 to 200 nucleotides. Telomeres can switch stochastically between two states: capped and uncapped. Capping is functionally defined as preserving the physical integrity of the chromosomes. To explain this protection, the T-loop/D-loop model has been proposed. In this model, the 3'G single-stranded overhang is tucked back inside the double-stranded telomeric DNA resulting in a large T-loop that is stabilized by an intramolecular D-loop. Formation of these loops involved a lots of proteins, which are directly or indirectly bound to telomeric DNA. Two proteins, called TRF1 and TRF2, seem to have a very important role in the formation of the loops. However, the molecular mechanism of such T-loop/D-loop is speculative, especially the process that involves TRF2 protein.The work presented in this thesis reports the NMR studies of the DNA binding domain of the TRF2 protein(Myb domain) and its interactions with telomeric DNA, following by the studies of 4 D-loop models.After the determination of the structure and internal dlmamics studies of Myb-TRF2 domain, studies of two complexes involving Myb-TRF2 protein and telomeric DNA are presented. These studies allow us to have a better understànding of Myb-TRF2 role and show that this protein has a sequence specifity and binds only double stranded-DNA. Then, 4 different D-loop models have been studied by NMR to determine their three-dimensional structures.Les télomères sont des complexes nucléoprotéiques localisés à l'extrémité des chromosomes des celluleseucaryotes. L'ADN télomérique humain contient entre 500 et 3000 repétitions de la séquence d(T2AG3)n (brin-G) et de son brin complémentaire C3TA2 (brin-C). Le brin G est terminé par une extrémité simple brin contenant de 50 à 200 nucléotides. Les télomères jouent des rôles essentiels au niveau cellulaire en permnettant la protection des chromosomes.Un modèle proposé pour expliquer le rôle protecteur des télomères est le modèle de la boucle-T (télomérique) / boucle-D (déplacement). Dans ce modèle, on observe le repliement de I'ADN télomérique double brin et I'insertion de la partie simple brin dans la partie double brin. La formation de ces boucles fait intervenir, directement ou indirectement, denombreuses protéines comme les protéines TRF1 et TRF2. Néanmoins les mécanismes moléculaires et notamment le rôle de la protéine TRF2 dans la formation de ces boucles restent spéculatifs.Le travail de thèse présenté dans ce manuscrit expose l'étude par Résonance Magnétique Nucléaire du domainede fixation à I'ADN de la protéine TRF2 (domaine Myb) et de ses interactions avec-l'ADN télomérique ainsi quel'étude de modèles de boucles-D.Après la détermination de la structure tridimensionnelle et l'étude de la dynamique interne du domaine Myb deTRF2, nous avons réalisé l'étude des interactions du domaine Myb avec I'ADN télomérique. Ainsi, deux complexes entre le domaine Myb de TRF2 et I'ADN télomérique ont été étudiés par RMN et ont permis de mieux comprendre le rôle du domaine Myb en montrant sa grande spécificité pour I'ADN télômérique double brin. Ensuite quatre modèles de boucles D différents ont été étudiés par RMN afin d'en déterminer les structures tridimensionnelles

Etude de complexes télomériques par résonance magnétique nucléaire

Les télomères sont des complexes nucléoprotéiques localisés à l extrémité des chromosomes des cellules eucaryotes. L ADN télomérique humain contient entre 500 et 3000 répétitions de la séquence d(T2AG3)n (brin-G) et de son brin complémentaire C3TA2 (brin-C). Le brin G est terminé par une extrémité simple brin contenant de 50 à 200 nucléotides. Les télomères jouent des rôles essentiels au niveau cellulaire en permettant la protection des chromosomes. Un modèle proposé pour expliquer le rôle protecteur des télomères est le modèle de la boucle-T (télomérique) / boucle-D (déplacement). Dans ce modèle, on observe le repliement de l ADN télomérique double brin et l insertion de la partie simple brin dans la partie double brin. La formation de ces boucles fait intervenir, directement ou indirectement, de nombreuses protéines comme les protéines TRF1 et TRF2. Néanmoins les mécanismes moléculaires et notamment le rôle de la protéine TRF2 dans la formation de ces boucles restent spéculatifs. Le travail de thèse présenté dans ce manuscrit expose l étude par Résonance Magnétique Nucléaire du domaine de fixation à l ADN de la protéine TRF2 (domaine Myb) et de ses interactions avec l ADN télomérique ainsi que l étude de modèles de boucles-D. Après la détermination de la structure tridimensionnelle et l étude de la dynamique interne du domaine Myb de TRF2, nous avons réalisé l étude des interactions du domaine Myb avec l ADN télomérique. Ainsi, deux complexes entre le domaine Myb de TRF2 et l ADN télomérique ont été étudiés par RMN et ont permis de mieux comprendre le rôle du domaine Myb en montrant sa grande spécificité pour l ADN télomérique double brin. Ensuite quatre modèles de boucles D différents ont été étudiés par RMN afin d en déterminer les structures tridimensionnelles.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis

The structure of human tRNALys3 anticodon bound to HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs