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NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome.

Two positive-strand HIV-1 genomic RNAs form a dimer in virion particles through interaction of the dimerization initiation sites (DIS). The DIS RNA fragment spontaneously formed a "loose-dimer" and was converted into a "tight-dimer" by supplementation with nucleocapsid protein NCp7. This two-step dimerization reaction requires the whole DIS sequence [Takahashi et al. (2000) RNA 6, 96-102]. In the present study, we measured imino proton resonances to investigate the secondary structures of the two types of dimers in a 39-mer RNA covering the entire DIS (DIS39), including discrimination between intra- and inter-molecular base pairing. Both the presence and absence of inter-molecular NOE between (15)N-labeled and unlabeled DIS39 were unambiguously detected in an equimolar mixture of (15)N-labeled and unlabeled DIS39. The stem-bulge-stem structures in both dimers were confirmed and found to be very close to each other from clear superimposition of the NMR spectra in the two dimeric states. Nevertheless, the modes of base pairing in the stems of the loose- and tight-dimers were intra- and inter-molecular, respectively. Our results suggest a large structural alteration of genomic RNA occurs during virion maturation.