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

Abstract

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...

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

“…Although not as favorable energetically as the formation of Watson-Crick base pairs, C⅐T, C⅐A, and G⅐T pairs (43)(44) can form between the (CAG) 10 and modified (CTG) 10 hairpins. As a third possibility, others have suggested that hairpin melting may occur prior to duplex conversion (25); in this scenario, the unstructured sequences would then anneal to form duplex. However, as monitored by optical methods, for all the hairpin pairs utilized in our experiments the temperature at which duplex formation occurs is well below the T m of the individual hairpins.…”

“…Similar methodology has been employed effectively to characterize the folded structures adopted by nucleic acids, including guanine quadruplexes (35), duplex and triplex DNA (36), and hairpin DNA (37) and RNA (25). In this work, we designed the FL-(CAG) 10 -DCL molecular beacon to selectively monitor the behavior of the (CAG) 10 hairpin in the presence of the complementary sequence.…”

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

“…2; 3; 7; 18-20 Experiments performed in vitro have shown that the metastable KL complex can subsequently isomerize into a more stable extended duplex (ED) that involves extensive intermolecular base-pairing along the entire SL1 sequence (Scheme 1). [21][22][23][24][25] The transition between KL and ED forms is catalyzed in vitro by NC, 23; 26-29 in a process that could explain the increased thermal stability observed for genomic RNA of HIV-1 and Moloney murine leukemia virus upon protease-dependent viral maturation. 30-32 The 3D structures of SL1 constructs in monomeric, 33-35 KL, 36-40 and ED form 38; 41-43 support the two-step dimerization model.…”

Effects of N2,N2-dimethylguanosine on RNA structure and stability: Crystal structure of an RNA duplex with tandem m22G:A pairs

“…In addition, hyperchromicity and the shape of the melting profiles provided further hints as to the (co)existence of hairpin and duplex secondary structures in solution. We are aware that for the quantification of hairpin/duplex ratios, other methods such as gel-shift assays under native conditions (Bernacchi et al 2005Ennifar et al 2007;Sun et al 2007) or NMR spectroscopy are required (Micura et al 2001). Here, the set of RNAs was exclusively investigated by UV absorbance/temperature profiles.…”