NMR-based identification of peptides that specifically recognize the d-arm of tRNA.

Human tRNA3(Lys) is used by HIV virus as a primer for the reverse transcription of its genome. The 18 nucleotides at the 3'-end of the tRNA3(Lys) are hybridized to a complementary sequence of the viral RNA called the primer-binding site. A screen against the human tRNA3(Lys) over a peptide library designed to target RNA has been performed. Of the 175 hexapeptides tested, three were found to bind to the d-stem of tRNA3(Lys). Alanine-scanning was used to define the determinants of the interaction between the peptides and tRNA3(Lys). They also bind to two other tested tRNAs, also at the level of the d-stem and loop, although the nucleotide sequence of the stem differs in one of them. These short peptides thus recognize specific structural features within the d-stem and loop of tRNAs. Associated with other pharmacophores, they could be useful to design optimized ligands targeting specific tRNAs such as retroviral replication primers

NMR structure of the Aquifex aeolicus tmRNA pseudoknot PK1: new insights into the recoding event of the ribosomal trans-translation

The transfer-messenger RNA (tmRNA) pseudoknot PK1 is essential for bacterial trans-translation, a ribosomal rescue mechanism. We report the solution structure of PK1 from Aquifex aeolicus, which despite an unprecedented small number of nucleotides and thus an unprecented compact size, displays a very high thermal stability. Several unusual structural features account for these properties and indicate that PK1 belongs to the class of ribosomal frameshift pseudoknots. This suggests a similarity between the mechanism of programmed ribosomal frameshifting and trans-translation

Optimizing HSQC experiment for the observation of exchange broadened signals in RNA–protein complexes

International audienceSites for interaction in protein–RNA complexes are often regions of conformational exchange. Although the study of exchange processes could bring valuable information about the recognition mode between protein and RNA, chemical exchange can be detrimental to the NMR spectra quality, resulting in broad, very weak or even unobservable signals. In the present report, we used CPMG–like experiments to improve HSQC spectra of an RNA–protein complex in fast exchange on the chemical shift time scale. The use of such improvement will allow us to handle the resolution of the three-dimensional structure of the complex by NMR

Synthèse d'analogues d'aminoglycosides par voie chimique et ingénierie métabolique (Application à l'étude des ARN par RMN du fluor)

Les ARN constituent des cibles thérapeutiques extrêmement intéressantes bien qu encore assez peu exploitées. En effet, les obstacles pour la conception de ligands spécifiques de ces cibles non traditionnelles, polyanioniques et très flexibles, sont encore loin d être levés. Les aminoglycosides, utilisés depuis longtemps pour leurs propriétés antibiotiques, sont souvent décrits comme des ligands universels d ARN. Leur structure constitue donc une architecture favorable pour l élaboration de nouveaux ligands spécifiques des ARN.Le but de cette thèse a été de développer une méthode systémique originale combinant chimie organique et microbiologie pour synthétiser de nouvelles molécules de structure analogue aux aminoglycosides, se fixant de façon spécifique sur des cibles ARN. Ce travail repose sur la compréhension récente des voies de biosynthèse des aminoglycosides permettant leur ingénierie rationnelle selon une stratégie de mutasynthèse. Cette approche expérimentale s appuie sur la conception de mimes de métabolites naturels pouvant être transformés par des bactéries génétiquement modifiées. Le développement de méthodologies novatrices en ingénierie métabolique, synthèse organique et chimie analytique nous a permis de concevoir des analogues d aminoglycosides fluorés qui se sont avérées être d excellentes sondes dans l étude des ARN par RMN du fluor.Pas de résumé en anglaisPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

International audienceTransfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA

NMR identification of ligands of aminoglycoside resistance enzymes

Bacterial resistance to aminoglycosides is mainly the result of enzyme-catalyzed chemical modifications of these antibiotics, which prevents their binding to their target. In order to circumvent this mechanism, an attractive possibility would be to block these enzymes, using selective inhibitors. This work describes a rational strategy aimed at isolating specific ligands of these enzymes, using NMR spectroscopy. Using magnetization transfer techniques, the identification of contacts between elementary pharmacophores and the protein target allows the guidance of hit improvement from a very early stage

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis.

Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N-Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N-Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent-exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context-dependent effect resulting from an interaction between neighboring charged side-chains

Spectral function of the 1D Hubbard model in the U→+∞U\to +\infty limit

We show that the one-particle spectral functions of the one-dimensional Hubbard model diverge at the Fermi energy like $|\omega-\varepsilon_F|^{-3/8}$ in the $U\to +\infty$ limit. The Luttinger liquid behaviour $|\omega-\varepsilon_F|^\alpha$, where $\alpha \to 1/8$ as $U\to +\infty$, should be limited to $|\omega-\varepsilon_F| \sim t^2/U$ (for $U$ large but finite), which shrinks to a single point, $\omega=\varepsilon_F$,in that limit. The consequences for the observation of the Luttinger liquid behaviour in photoemission and inverse photoemission experiments are discussed.Comment: 4 pages, RevTeX, 2 figures on reques

Phase diagram of the one-dimensional extended Hubbard model with attractive and/or repulsive interactions at quarter filling

We study the phase diagram of the one dimensional (1D) $U-V$ model at quarter filling in the most general case where the on-site and first-neighbour interactions $U$ and $V$ can be both attractive and repulsive. The results have been obtained using exact diagonalization of small clusters and variational techniques, as well as exact results in various limits. We have analyzed four properties of the groundstate: i)~whether it is insulating or metallic; \hbox{ii)~whether} it is homogenous or phase separated; iii)~whether it has a spin gap; iv)~whether it has dominant superconducting fluctuations. With eight phases, the resulting phase diagram is unexpectedly rich. The four phases not found in the weak coupling limit are: i) an insulating phase when $U$ and $V$ are large enough; ii) a region of phase separation when $V$ is attractive; iii) another region of phase separation when $V$ is large enough and $U$ small; iv) a region with dominant superconducting fluctuations when $V$ is intermediate and $U$ small. The actual nature of this last phase, which has pairs but no spin gap, is not fully clear yet.Comment: 24 pages, RevTeX (4 postscript figures attached to the end