The catalytic triad of rice NARROW LEAF1 involves H234.

NARROW LEAF1 (NAL1) exerts a multifaceted influence on leaf morphology and crop yield. Recent crystal study proposed that histidine 233 (H233) is part of the catalytic triad. Here we report that unlike suggested previously, H234 instead of H233 is a component of the catalytic triad alongside residues D291 and S385 in NAL1.

Retroviral nucleocapsid proteins and DNA strand transfers.

An infectious retroviral particle contains 1000-1500 molecules of the nucleocapsid protein (NC) that cover the diploid RNA genome. NC is a small zinc finger protein that possesses nucleic acid chaperone activity that enables NC to rearrange DNA and RNA molecules into the most thermodynamically stable structures usually those containing the maximum number of base pairs. Thanks to the chaperone activity, NC plays an essential role in reverse transcription of the retroviral genome by facilitating the strand transfer reactions of this process.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations.

Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex.

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out.

Identification of acylation products in SHAPE chemistry.

SHAPE chemistry (selective 2'-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2'-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC-MS/MS experiments.

Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding.

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing.

RNA SHAPE chemistry with aromatic acylating reagents.

As chemical methods for RNA secondary structure determination, SHAPE chemistry (selective 2'-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature. In order to improve the specificity of acylating reagents towards unpaired nucleotides, we have explored the reactivity of symmetric anhydrides, acyl fluorides, active esters like succinimidyl ester and cyanomethyl esters for 2'-O-acylation reaction. Among the tested compounds, only the acyl fluoride 4 showed a low reactivity (compared to NMIA).

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1).

Dynamic interactions of the HIV-1 Tat with nucleic acids are critical for Tat activity in reverse transcription.

The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer.

Intrinsic nucleic acid dynamics modulates HIV-1 nucleocapsid protein binding to its targets.

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem.

Flexible nature and specific functions of the HIV-1 nucleocapsid protein.

One salient feature of reverse transcription in retroviruses, notably in the human immunodeficiency virus type 1, is that it requires the homologous nucleocapsid (NC) protein acting as a chaperoning partner of the genomic RNA template and the reverse transcriptase, from the initiation to the completion of viral DNA synthesis. This short review on the NC protein of human immunodeficiency virus type 1 aims at briefly presenting the flexible nature of NC protein, how it interacts with nucleic acids via its invariant zinc fingers and flanking basic residues, and the possible mechanisms that account for its multiple functions in the early steps of virus replication, notably in the obligatory strand transfer reactions during viral DNA synthesis by the reverse transcriptase enzyme.

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.

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).

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).

Deciphering the complete mitochondrial genome and phylogeny of the extinct cave bear in the Paleolithic painted cave of Chauvet.

Retrieving a large amount of genetic information from extinct species was demonstrated feasible, but complete mitochondrial genome sequences have only been deciphered for the moa, a bird that became extinct a few hundred years ago, and for Pleistocene species, such as the woolly mammoth and the mastodon, both of which could be studied from animals embedded in permafrost. To enlarge the diversity of mitochondrial genomes available for Pleistocene species, we turned to the cave bear (Ursus spelaeus), whose only remains consist of skeletal elements. We collected bone samples from the Paleolithic painted cave of Chauvet-Pont d'Arc (France), which displays the earliest known human drawings, and contains thousands of bear remains.

Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA.

The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one.

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C.

Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer.

In vitro characterization of a base pairing interaction between the primer binding site and the minimal packaging signal of avian leukosis virus genomic RNA.

The 5' leader region of avian sarcoma-leukosis viruses (ASLVs) folds into a series of RNA secondary structures which are involved in key steps in the viral replication cycle such as reverse transcription, dimerization and packaging of genomic RNA. The O3 stem and three stem-loops (O3SLa, O3SLb and O3SLc) form the minimal packaging signal that is located downstream of the primer binding site (PBS). The U5-PBS region contributes to packaging via a mechanism that remains unknown.