Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer

Takahashi, Keníchi; Baba, Seiki; Koyanagi, Yoshio; Yamamoto, Naoki; Takaku, Hiroshi; Kawai, Gota

doi:10.1074/jbc.m104577200

Cited by 32 publications

(26 citation statements)

References 45 publications

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“…Many studies have been carried out to investigate the DIS kissing complex to extended duplex transition, either on a similar 23-nucleotide fragment or on larger fragments comprising the complete 39-nucleotide DIS stem-loop (17,28,34,(45)(46)(47)(48)(49)(50)(51)(52). Notably, the chaperone activity of the NCp7 was shown to facilitate this transition (17).…”

Section: Discussionmentioning

confidence: 99%

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: 99%

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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“…A second way in which HIV-1 and HIV-2 RNAs behave differently is that the HIV-2 leader RNA fragment was unable to form tight dimers (16,17), contrary to HIV-1 RNA (10,11,18). These exceptionally stable dimers could withstand semi-denaturing electrophoresis in TBE buffer at 24°C.…”

Section: Introductionmentioning

confidence: 99%

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

et al. 2003

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An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1 a 5′ leader region site referred to as stem-loop1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5′-end of the primerbinding site (PBS) and a stem loop homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization/electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5′ and/or 3′ ends and by making compensatory base substitutions we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1- † This research was supported by the National Institutes of Health grant number AI45388 to J.S.L.

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“…[16][17][18] At the end, the ESI-FTICR spectrum obtained from wild-type SL1A contained familiar monomeric and dimeric species, together with a previously absent 4:2 complex (Figure 6a). The conformational state of all dimeric assemblies in the sample was again interrogated by mild SORI-CID, which revealed very distinctive dissociation patterns.…”

Section: Nc-hinge Interactions and Isomerization Efficiencymentioning

confidence: 99%

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

Turner

Hagan

Fabris

2007

Journal of Molecular Biology

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The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5′-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3′-end. This binding mode is supported by the presence of a grip-like motif described by recent crystal structures, which is formed by the 5′-purines of both hairpins held together by mutual stacking interactions. Using tandem mass spectrometry, hinge interactions were clearly shown to reduce the efficiency of KL/ED isomerization without inducing its complete block. This outcome is consistent with the partial stabilization of the extra-helical grip by the bound protein, which can hamper the purine components from parting ways and initiate the strand exchange process. These findings confirm that the broad binding and chaperone activities of NC induce unique effects that are clearly dependent on the structural context of the cognate nucleic acid substrate. For this reason, the presence of multiple binding sites on the different forms assumed by SL1 can produce seemingly contrasting effects that contribute to a fine modulation of the two-step process of RNA dimerization and isomerization.

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Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer

Cited by 32 publications

References 45 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

Elements Located Upstream and Downstream of the Major Splice Donor Site Influence the Ability of HIV-2 Leader RNA To Dimerize in Vitro

Understanding the Isomerization of the HIV-1 Dimerization Initiation Domain by the Nucleocapsid Protein

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