Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids

Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA–DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop–loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop–loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open ‘Y’ conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the ‘Y’ conformation exhibiting at least 12 unpaired nucleotides in its lower part

RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC

General survey of hAT transposon superfamily with highlight on hobo element in Drosophila

International audienceThe hAT transposons, very abundant in all kingdoms, have a common evolutionary origin probably predating the plant-fungi-animal divergence. In this paper we present their general characteristics. Members of this superfamily belong to Class II transposable elements. hAT elements share transposase, short terminal inverted repeats and eight base-pairs duplication of genomic target. We focus on hAT elements in Drosophila, especially hobo. Its distribution, dynamics and impact on genome restructuring in laboratory strains as well as in natural populations are reported. Finally, the evolutionary history of hAT elements, their domestication and use as transgenic tools are discussed

Determination and Rietveld refinement of the crystal structure of Li0.50Ni0.25TiO(PO4) from powder x-ray and neutron diffraction

The structure of the oxyphosphate Li0.50Ni0.25TiO(PO4) has been determined from conventional X-ray and neutron powder diffraction data. The parameters of the monoclinic cell (space group P21/c, Z=4), obtained from X-ray results, are: a=6.3954(6) Å, b=7.2599(6) Å, c=7.3700(5) Å, and β=90.266(6)°; those resulting from neutron study are: a=6.3906(7) Å, b=7.2568(7) Å, c=7.3673(9) Å, and β=90.234(7)°. Refinement by the Rietveld method using whole profile, leads to satisfactory reliability factors: cRwp=0.128, cRp=0.100, and RB=0.038 for X-ray and cRwp=0.110, cRp=0.120, and RB=0.060 for neutrons. The structure of Li0.50Ni0.25TiO(PO4) can be described as a TiOPO4 framework constituted by chains of tilted corner-sharing TiO6 octahedra running parallel to the c axis and cross linked by phosphate tetrahedra. In this framework, there are octahedral cavities occupied by Li and Ni atoms: Li occupies the totality of the 2a sites and Ni occupies statistically half of the 2b sites. Ti atoms are displaced from the center of octahedra units in alternating long (2.242 Å) and short (1.711 Å) Ti-O bonds along chains

New process of preparation, X-ray characterisation, structure and vibrational studies of a solid solution LiTiOAs₁₋<i>_x</i>P<i>_x</i>O₄ (0 ≤ x ≤ 1)

LiTiOAs1−xPxO4 (0 ≤ x ≤ 1) compounds have been prepared using solutions of Li, Ti, As and P elements as starting products. Selected compositions have been investigated by powder X-ray or neutrons diffraction analysis, Raman and infrared spectroscopy. The structure of LiTiOAs1−xPxO4 (x=0, 0.5 and 1) samples determined by Rietveld analysis is orthorhombic with Pnma space group. It is formed by a 3D network of TiO6 octahedra and XO4 (X=As1−xPx) tetrahedra where octahedral cavities are occupied by lithium atoms. TiO6 octahedra are linked together by corners and form infinite chains along a-axis. Ti atoms are displaced from the centre of octahedral units in alternating short (1.700–1.709 Å) and long (2.301–2.275 Å) Ti–O bonds. Raman and infrared studies confirm the existence of Ti–O–Ti chains. Thermal stability of LiTiOAsO4 has been reported

Crystal structure and spectroscopic properties of a new oxyarsenate Li0.5Ni0.25TiOAsO4

The new oxyarsenate Li0.5Ni0.25TiOAsO4 has been synthesized and studied by a combination of X-ray powder diffraction, neutrons powder diffraction and vibrational spectroscopy. Li0.5Ni0.25TiOAsO4 crystallizes in the monoclinic P21/c space group with the unit cell parameters..

New process of preparation, X-ray characterisation, structure and vibrational studies of a solid solution LiTiOAs₁₋<i>_x</i>P<i>_x</i>O₄ (0 ≤ x ≤ 1)

LiTiOAs1−xPxO4 (0 ≤ x ≤ 1) compounds have been prepared using solutions of Li, Ti, As and P elements as starting products. Selected compositions have been investigated by powder X-ray or neutrons diffraction analysis, Raman and infrared spectroscopy. The structure of LiTiOAs1−xPxO4 (x=0, 0.5 and 1) samples determined by Rietveld analysis is orthorhombic with Pnma space group. It is formed by a 3D network of TiO6 octahedra and XO4 (X=As1−xPx) tetrahedra where octahedral cavities are occupied by lithium atoms. TiO6 octahedra are linked together by corners and form infinite chains along a-axis. Ti atoms are displaced from the centre of octahedral units in alternating short (1.700–1.709 Å) and long (2.301–2.275 Å) Ti–O bonds. Raman and infrared studies confirm the existence of Ti–O–Ti chains. Thermal stability of LiTiOAsO4 has been reported

Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin. Nucleic Acids Res

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction