Structure of the dimer a initiation complex of HIV-1 genomic RNA

Mujeeb, Anwer; Clever, Jared L.; Billeci, Todd M.; James, Thomas Leroy; Parslow, Tristram G.

doi:10.1038/nsb0698-432

Cited by 160 publications

(180 citation statements)

References 26 publications

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“…Indeed, many structural studies relied on a flash cooling procedure to ensure the formation of the hairpin form. This was the case for several crystallographic studies (see Introduction) as well as for an NMR study of the DIS(Lai) kissing complex (44). More importantly, most of the crystal studies performed for describing a hairpin structure finally resulted in the description of a duplex structure, which clearly shows that the cooling procedure was unimportant.…”

Section: Discussionmentioning

confidence: 96%

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

Bernacchi

Ennifar

Tóth

et al. 2005

Journal of Biological Chemistry

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We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 M in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 M in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.Stem-loops and bulged loops are common motifs found in RNA secondary structure and are often involved in RNA-protein or RNA-RNA recognition. Because of the self-complementarity of stems, a stem-loop structure can also adopt an alternative duplex structure containing unpaired nucleotides. This hairpin-duplex equilibrium also represents a major problem in structural studies of nucleic acids by NMR and x-ray crystallography, both requiring concentrated samples. Strategies using a mixture of unlabeled and 15 N-labeled oligonucleotides were developed to discriminate between the two species by NMR (1, 2). However, many attempts at crystallizing RNA or DNA stem-loop structures led unexpectedly to duplex crystal structures (3-6). This was even the case for the unusually stable RNA tetraloops belonging to the GNRA, UNCG, and CUUG families, which are supposed to be nucleation points for RNA folding because of their stability and quick folding kinetics (see "Discussion" in Ref. 7). Significantly, this problem does not seem to spread to NMR studies because several RNA stem-loop structures were successfully solved by using this technique, whereas most of the hairpin structures described by x-ray crystallography were obtained from complexes with proteins (8 -10) or were embedded in a larger RNA context, as for tRNAs, hammerhead ribozyme, and ribosome structures. The only notable exception is the GAGA tetraloop found in the sarcin-ricin loop, successfully crystallized as a hairpin (11,12).The present study focuses on the dimerization initiation site (DIS) 4 of HIV-1 genomic RNA that has been extensively studied by several biochemical and biophysical methods. This strongly conserved stem-loop sequence (13) (Fig. 1a) was shown to be cruc...

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Section: Discussionmentioning

confidence: 96%

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

Bernacchi

Ennifar

Tóth

et al. 2005

Journal of Biological Chemistry

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“…In these complexes, base complementarity exists not only between the intermolecular hairpin loops, but also between the stem regions. In the case of the dimer initiation complex of the HIV-genomic RNA, a metastable intermolecular kissing interaction culminates in a linear duplex isoform stabilized by intermolecular base pairing (20,44). A kissing interaction also initiates the binding of the antisense RNA CopA to its target, CopT, but the outcome of this interaction is an unusual four-way junction defined by the predicted base stacking arrangements of intramolecular helices and newly formed intermolecular helices (19,45).…”

Section: Discussionmentioning

confidence: 99%

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Andersen

Collins

2001

Proc. Natl. Acad. Sci. U.S.A.

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Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra-and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stemloops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.R NA kissing interactions, also called loop-loop pseudoknots, occur when the unpaired nucleotides in one hairpin loop base pair with the unpaired nucleotides in another hairpin loop (1). When the hairpin loops are located on separate RNA molecules, their intermolecular interaction is called a kissing complex. These interactions generally form between stem-loops containing extensive complementarity; however, stable complexes have been observed containing only two intermolecular Watson-Crick base pairs (2).Intramolecular kissing interactions are observed in the native structures of a variety of RNAs including Varkud satellite (VS) RNA, tRNA, and group I introns (3-6). These kissing interactions contribute to the assembly and stabilization of their respective RNA structures by joining and orienting helices. Kissing interactions may stabilize both native and nonnative interactions during tertiary folding, which can affect the rate at which the native structure is formed (7). Kissing interactions often form distorted structures that can serve as recognition sites for proteins, RNAs, metal ions, or other ligands (8-10). As a result, kissing interactions contribute to the stability of an RNA structure by affecting both global and local RNA interactions.The transient formation of an intermolecular kissing complex is required for RNA dimerization during the life cycle of retroviruses (11-15), and for the formation of some antisensetarget complexes (16,17). Kissing complexes in which the loop nucleotides are complementary can form stable dimers that contain intermolecular base pairs between the loop nucleotides only. Other stem-loops with more extensive complementarity sometimes form unstable kissing complexes, which are quickly remodeled into stable duplex or cruciform isoforms (18)(19)(20). Secondary structure remodeling involves breaking intramolecular base pairs in each hairpin and using the bases to form intermolecular helices.The VS ribozyme contains a kissing interaction that is required for self-cleavage of the wild-type RNA in vitro (6). This interaction involves Watson-Crick bas...

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“…NMR data recorded on weakly aligned samples allowed us to obtain a structural model in solution of an RNA-RNA kissing complex with higher accuracy and precision than previously reported kissing complex solution structures (14,20,(22)(23)(24)(25)(26). This structural study revealed the key atomic interactions responsible for the stabilizing role of the GA base pair at the loop-closing position of kissing complexes.…”

mentioning

confidence: 88%

“…First, the number of paired loop residues can vary from two up to seven (22,23,35). In addition, some of the kissing complexes possess nonpaired bases (22,24,25,(47)(48)(49). For example, the crystal structures of the HIV-1 MAL and LAI kissing complexes (49), the anticodon-anticodon interaction in tRNA Asp (47), and a loop-loop interaction in 23S rRNA (48) show coaxial alignment of the two opposite stems due to flanking residues on the 5Ј side.…”

Section: Curvature Of Kissing Complexes Lacking Unpaired Residuesmentioning

confidence: 99%

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

Melckebeke

Devany

Primo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Structure of the dimer a initiation complex of HIV-1 genomic RNA

Cited by 160 publications

References 26 publications

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

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